FDM 3D-Printed Sustained-Release Gastric-Floating Verapamil Hydrochloride Formulations with Cylinder, Capsule and Hemisphere Shapes, and Low Infill Percentage

The aim of this work was to design and fabricate fused deposition modeling (FDM) 3D-printed sustained-release gastric-floating formulations with different shapes (cylinder, capsule and hemisphere) and infill percentages (0% and 15%), and to investigate the influence of shape and infill percentage on the properties of the printed formulations. Drug-loaded filaments containing HPMC, Soluplus® and verapamil hydrochloride were prepared via hot-melt extrusion (HME) and then used to print the following gastric-floating formulations: cylinder-15, capsule-0, capsule-15, hemisphere-0 and hemisphere-15. The morphology of the filaments and the printed formulations were observed by scanning electron microscopy (SEM). The physical state of the drugs in the filaments and the printed formulations were characterized by X-ray diffraction (XRD), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The printed formulations were evaluated in vitro, including the weight variation, hardness, floating time, drug content and drug release. The results showed that the drug-loaded filament prepared was successful in printing the gastric floating formulations. Verapamil hydrochloride was proved thermally stable during HME and FDM, and in an amorphous state in the filament and the printed formulations. The shape and infill percentage of the printed formulations effected the hardness, floating time and in vitro drug release.


Introduction
Oral administration is a preferred drug delivery route for its high patient compliance, low cost, non-invasiveness and ease of use [1]. However, the bioavailability of conventional oral dosage forms may be influenced by short gastro-retentive time or unpredictable gastric emptying times, especially for drugs which are absorbed in the upper part of the digestive system, or more soluble in acidic environments [2]. To solve this problem, a series of gastroretentive systems, including mucoadhesion systems, expandable systems and floating systems, were developed to prolong gastric residence time of drugs to obtain better drug absorption and bioavailability [3,4]. Among all these gastroretentive systems, the gastrofloating systems attracted special attention due to their low density that allowed them to float on gastric fluids without affecting the gastric emptying rate [4]. Traditionally, gastrofloating systems rely on swelling polymers, microporous components or gas-generating systems to achieve low density, which usually leads to their complex composition and difficulty in manufacturing [4,5]. In addition, the gastro-floating systems are sometimes

Preparation of Filaments
Filaments were prepared using the HME method. Beforehand, homogeneous mixing of the excipients and drug was obtained by the incremental method and thoroughly grinded with a mortar and pestle. To prepare the drug-loaded filaments, the verapamil hydrochloride, HPMC, Soluplus ® and PEG 400 were mixed homogeneously at a weight ratio of 10:42.5:42.5:5. The mixture was then extruded by a single-screw hot-melt extruder (FilaBot ® FOV1, USA) at 115 °C. The nozzle diameter of the extruder was 1.7 ± 0.1 mm. Blank filaments were also prepared by HME. The composition of blank filaments was HPMC, Soluplus ® and PEG 400 at a weight ratio of 47.2:47.2:5.6 (close to 42.5:42.5:5). The extruding condition was the same as that of the drug-loaded filaments.

3D Printing of Gastric-Floating Formulations
The drug-loaded filaments were used to fabricate the designed formulations by a desktop 3D printer (MakerBot Replicator 2X, MakerBot, New York, NY, USA). The temperatures for the nozzle and building platform were 195 °C and 90 °C, respectively. The speed of extruding and traveling for the printing head was 90-100 mm/s and 150 mm/s, respectively.

Preparation of Filaments
Filaments were prepared using the HME method. Beforehand, homogeneous mixing of the excipients and drug was obtained by the incremental method and thoroughly grinded with a mortar and pestle. To prepare the drug-loaded filaments, the verapamil hydrochloride, HPMC, Soluplus ® and PEG 400 were mixed homogeneously at a weight ratio of 10:42.5:42.5:5. The mixture was then extruded by a single-screw hot-melt extruder (FilaBot ® FOV1, USA) at 115 • C. The nozzle diameter of the extruder was 1.7 ± 0.1 mm. Blank filaments were also prepared by HME. The composition of blank filaments was HPMC, Soluplus ® and PEG 400 at a weight ratio of 47.2:47.2:5.6 (close to 42.5:42.5:5). The extruding condition was the same as that of the drug-loaded filaments.

3D Printing of Gastric-Floating Formulations
The drug-loaded filaments were used to fabricate the designed formulations by a desktop 3D printer (MakerBot Replicator 2X, MakerBot, New York, NY, USA). The temperatures for the nozzle and building platform were 195 • C and 90 • C, respectively. The speed of extruding and traveling for the printing head was 90-100 mm/s and 150 mm/s, respectively.

Morphology
Photographs of the blank filaments, drug-loaded filaments and 3D-printed formulations were taken with a digital camera (Canon IXUS 220 HS, Tokyo, Japan). The surface and section images of the blank filaments, drug-loaded filaments and 3D-printed formulations (at magnifications 30× and 1000×) were separately obtained by a scanning electron microscopy (SEM, JSM-IT300 Scanning Microscope, JEOL, Tokyo, Japan) at an accelerating voltage of 5.0 kV.

X-ray Powder Diffraction
X-ray powder diffraction (XRD, Mini Flex 600, Rigaku, Japan) was performed to assess the physical state of verapamil hydrochloride in the drug-loaded filaments and formulations. The analysis was carried out using a Cu X-ray source, and the operating current and voltage were set at 15 mA and 40 kV, respectively. All samples including verapamil hydrochloride, HPMC, Soluplus ® , physical mixture (composed of verapamil hydrochloride, HPMC, Soluplus ® and PEG 400 at weight ratio of 10:42.5:42.5:5), drugloaded filaments and 3D-printed formulations were scanned from 2θ of 5 • to 60 • at a speed of 5 • /min [25][26][27].

Thermal Analysis
A thermogravimetric analyzer (Q600 SDT, TA Instruments, New Castle, DE, USA) was employed to assay the thermal stability of verapamil hydrochloride, HPMC, Soluplus ® , physical mixture, drug-loaded filaments and 3D-printed formulations. The samples (about 5 mg) were weighted accurately, loaded in ceramic pans, and heated from room temperature to 500 • C. The test was carried out at a heating rate of 10 • C/min under a nitrogen purge of 100 mL/min. The data were analyzed using TA 2000 analysis software [28,29].
Differential scanning calorimetry (DSC, Q100 DSC, TA Instruments, USA) analysis was also conducted. After being dried in a desiccator, samples (about 5 mg) were loaded on aluminum pans, and the test was performed at a heating rate of 10 • C/min under a nitrogen purge of 50 mL/min. The heat scan from −20 to 200 • C was conducted for verapamil hydrochloride, HPMC, Soluplus ® , physical mixture, drug-loaded filaments and 3D-printed formulations. Then, PEG 400 was scanned from −50 to 100 • C. Verapamil hydrochloride was assayed by a heat, quench and heat process. The data were analyzed using TA 2000 analysis software [30,31].

Weight
The 3D-printed formulations were weighed using an electronic analytical balance (ME104, Mettler Toledo, Zurich, Switzerland) (n = 10). The average weight and weight variances of the formulations were calculated, respectively.

Hardness
The hardness of 3D-printed formulations was measured with a texture analyzer (MultiTest 2.5-I, Mecmesin, Slinfold, UK). A vertical pressure was put on the formulations by a 75 mm diameter probe at a speed of 1 mm/s and the maximum force of 800 N. The measurements were performed at two mutually perpendicular directions for each formulation (illustrated as Figure 2) (n = 6), considering that the structure of 3D-printed formulations was anisotropic, compared with that of traditional pressed formulations [10].

In Vitro Floating Behavior
The in vitro floating capacity of the formulations was tested according to the previous literatures [32,33]. Then, 100 mL of 0.1 mol/L hydrochloric acid solution was added in a beaker as floating medium and kept at 37 ± 0.5 • C in a thermostatic water bath. Each of the 3D-printed formulations was put into the medium and the floating behavior of the formulation was monitored and recorded. The tests were performed in triplicate for each formulation (n = 3).

Drug Content
Samples of the drug-loaded filaments and 3D-printed formulations (approximately 0.4 g) were precisely weighed and separately dissolved in 0.1 mol/L hydrochloric acid solutions (n = 3). After a proper dilution, the drug content of each sample was determined by high-performance liquid chromatography (HPLC).
Agilent UV-HPLC 1260 series (Agilent Technologies, Germany) were employed with Agilent Zorbax Extended C18 column (150 mm × 4.6 mm, particle size 5 µm) maintained at Pharmaceutics 2022, 14, 281 5 of 14 40 • C. The mobile phase consisted of methanol and acetate buffer solution (pH was adjusted to 4.1 with triethylamine) at a volume ratio of 45:55. The flow rate of the mobile phase was 1.0 mL/min and the volume of sample injected was 20 µL. The analytical wavelength of the UV detector was set at 278 nm [21,22].

In Vitro Floating Behavior
The in vitro floating capacity of the formulations was tested according to the previous literatures [32,33]. Then, 100 mL of 0.1 mol/L hydrochloric acid solution was added in a beaker as floating medium and kept at 37 ± 0.5 °C in a thermostatic water bath. Each of the 3D-printed formulations was put into the medium and the floating behavior of the formulation was monitored and recorded. The tests were performed in triplicate for each formulation (n = 3).

Drug Content
Samples of the drug-loaded filaments and 3D-printed formulations (approximately 0.4 g) were precisely weighed and separately dissolved in 0.1 mol/L hydrochloric acid solutions (n = 3). After a proper dilution, the drug content of each sample was determined by high-performance liquid chromatography (HPLC).
Agilent UV-HPLC 1260 series (Agilent Technologies, Germany) were employed with Agilent Zorbax Extended C18 column (150 mm × 4.6 mm, particle size 5 µm) maintained at 40 °C. The mobile phase consisted of methanol and acetate buffer solution (pH was adjusted to 4.1 with triethylamine) at a volume ratio of 45:55. The flow rate of the mobile phase was 1.0 mL/min and the volume of sample injected was 20 µL. The analytical wavelength of the UV detector was set at 278 nm [21,22].

In Vitro Drug Release
The in vitro drug release of 3D-printed gastric-floating formulations was tested according to the United States Pharmacopeia (USP) dissolution apparatus I, and the commercial sustained-release verapamil hydrochloride tablet was used as a control (n = 6). 900 mL of 0.1 mol/L hydrochloric acid solution was added into each vessel and kept at 37.0 ± 0.5 °C with a stirring speed of 100 r/min [21,22]. 10 mL of the released medium was withdrawn at each predetermined time point, and replaced immediately with fresh medium

In Vitro Drug Release
The in vitro drug release of 3D-printed gastric-floating formulations was tested according to the United States Pharmacopeia (USP) dissolution apparatus I, and the commercial sustained-release verapamil hydrochloride tablet was used as a control (n = 6). 900 mL of 0.1 mol/L hydrochloric acid solution was added into each vessel and kept at 37.0 ± 0.5 • C with a stirring speed of 100 r/min [21,22]. 10 mL of the released medium was withdrawn at each predetermined time point, and replaced immediately with fresh medium at the same volume and temperature. All samples were filtered through a 0.2-µm filter and the concentrations of verapamil hydrochloride were assayed by HPLC at the same condition of drug content.

Statistical Analysis
In this study, t-test was employed using GraphPad Prism software (version 6.01) to analyze the results. Differences in results where p < 0.05 were considered significant, and p < 0.01 were considered extremely significant.

Morphology
The appearance and SEM photographs of the blank and drug-loaded filaments are shown in Figure 3. Both filaments showed smooth surface and homogeneous color ( Figure 3A,E). The blank filaments were more transparent than the drug-loaded filaments. In the low magnification (30×) SEM side view, the blank filaments ( Figure 3B) showed a smoother surface than drug-loaded filaments ( Figure 3F), and both blank and drug-loaded filaments ( Figure 3C,G) showed non-porous cross-sections. In the high magnification (1000×) side view, no crystal of the drug was observed on the surface of drug-loaded filaments ( Figure 3H) compared with that of blank filaments ( Figure 3D). shown in Figure 3. Both filaments showed smooth surface and homogeneous color ( Figure  3A,E). The blank filaments were more transparent than the drug-loaded filaments. In the low magnification (30×) SEM side view, the blank filaments ( Figure 3B) showed a smoother surface than drug-loaded filaments ( Figure 3F), and both blank and drugloaded filaments ( Figure 3C,G) showed non-porous cross-sections. In the high magnification (1000×) side view, no crystal of the drug was observed on the surface of drug-loaded filaments ( Figure 3H) compared with that of blank filaments ( Figure 3D).  As the temperature (195 • C) for printing formulations was higher than the melting point of verapamil hydrochloride (146 • C), the drug was supposed to disperse molecularly or in an amorphous state [23,34].  The appearance and SEM photographs of the cross-sections of the three formulations are further shown in Figure 5, presenting the inner structure of the printed formulations. Low infill percentage was designed for all formulations to ensure the feasibility of floating. When the infill percentage was set at 0% for cylinder formulations, the flat top tended to collapse, whereas capsule-0 and hemisphere-0 formulations were printed successfully by the effective support of arched structures. Thus, only 15% of the infill percentage was designed for the cylinder formulations (cylinder-15). In order to obtain comparable formulations among the three shapes of formulations, 15% of infill percentage was also utilized for capsule-15 and hemisphere-15. The appearance and SEM photographs of the cross-sections of the three formulations are further shown in Figure 5, presenting the inner structure of the printed formulations. Low infill percentage was designed for all formulations to ensure the feasibility of floating. When the infill percentage was set at 0% for cylinder formulations, the flat top tended to collapse, whereas capsule-0 and hemisphere-0 formulations were printed successfully by the effective support of arched structures. Thus, only 15% of the infill percentage was designed for the cylinder formulations (cylinder-15). In order to obtain comparable The appearance and SEM photographs of the cross-sections of the three formulations are further shown in Figure 5, presenting the inner structure of the printed formulations. Low infill percentage was designed for all formulations to ensure the feasibility of floating. When the infill percentage was set at 0% for cylinder formulations, the flat top tended to collapse, whereas capsule-0 and hemisphere-0 formulations were printed successfully by the effective support of arched structures. Thus, only 15% of the infill percentage was designed for the cylinder formulations (cylinder-15). In order to obtain comparable formulations among the three shapes of formulations, 15% of infill percentage was also utilized for capsule-15 and hemisphere-15.

X-ray Powder Diffraction
The result of XRD is shown in Figure 6. The pure verapamil hydrochloride showed several characteristic peaks at 2θ of 10

X-ray Powder Diffraction
The result of XRD is shown in Figure 6. The pure verapamil hydrochloride showed several characteristic peaks at 2θ of 10 [25,35]. The HPMC and Soluplus ® showed broad peaks with the peak of maximum intensity at 9.09 • and 18.06 • , respectively, which indicated their amorphous behavior. The diffraction peaks of verapamil hydrochloride could be found in the physical mixture but disappeared in the drug-loaded filaments and 3D-printing formulations, indicating that there was no drug crystal existing after HME and the 3D-printing process [36]. This was consistent with the SEM photograph in which no drug crystal was founded on either filament or formulation. showed broad peaks with the peak of maximum intensity at 9.09° and 18.06°, respectively, which indicated their amorphous behavior. The diffraction peaks of verapamil hydrochloride could be found in the physical mixture but disappeared in the drug-loaded filaments and 3D-printing formulations, indicating that there was no drug crystal existing after HME and the 3D-printing process [36]. This was consistent with the SEM photograph in which no drug crystal was founded on either filament or formulation.

Thermal Analysis
As shown in the Figure 7, the TGA result showed that the drug and excipients were all thermally stable at the temperature of HME (115 °C) and FDM printing (195 °C). All samples showed less than 5% weight loss at 195 °C, indicating their thermostability under

Thermal Analysis
As shown in the Figure 7, the TGA result showed that the drug and excipients were all thermally stable at the temperature of HME (115 • C) and FDM printing (195 • C). All samples showed less than 5% weight loss at 195 • C, indicating their thermostability under FDM condition. The weight loss under 60 • C was considered as attributing to be the evaporation of water. The hygroscopicity of Soluplus ® and HPMC was in accordance with the previous reports [37][38][39]. The DSC result is shown in Figure 8. An endothermic peak of verapamil hydrochloride showed a melting point at 145.9 °C, which was consistent with literature (138.5 °C~148.1 °C) [34]. An endothermic peak of physical mixture was also observed at 135.8 °C, indicating the existence of verapamil crystal. The decrease in melting point was supposed to be related with the plasticization of PEG 400. However, no obvious endothermic peak was observed in drug-loaded filaments or 3D-printing formulations, indicating that most of the drug did not exist in crystal form. The result was consistent with those of SEM and XRD. Combining the thermograms of verapamil hydrochloride (glass transition temperature (Tg) at 52.3 °C, Figure S1 in Supplementary Materials) and PEG 400 (melting point at 5 °C, Figure S2 in Supplementary Materials), the transitions of physical mixture, filament and formulations from 40 to 60 °C demonstrated that the amorphous drug existed and gradually became less in them. Still, the drug did not distribute molecularly homogenous in the formulations, which might have potential implications to the dissolution findings. Additionally, the Tg of pure Soluplus ® was at 78.4 °C, which was close to the previous report [40]. The Tg of Soluplus ® decreased successively in the order of physical mixture, filament and formulations, indicating the mixture was more homogeneous and the plasticization of PEG 400 was more effective. The DSC result is shown in Figure 8. An endothermic peak of verapamil hydrochloride showed a melting point at 145.9 • C, which was consistent with literature (138.5 • C~148.1 • C) [34]. An endothermic peak of physical mixture was also observed at 135.8 • C, indicating the existence of verapamil crystal. The decrease in melting point was supposed to be related with the plasticization of PEG 400. However, no obvious endothermic peak was observed in drug-loaded filaments or 3D-printing formulations, indicating that most of the drug did not exist in crystal form. The result was consistent with those of SEM and XRD. Combining the thermograms of verapamil hydrochloride (glass transition temperature (Tg) at 52.3 • C, Figure S1 in Supplementary Materials) and PEG 400 (melting point at 5 • C, Figure S2 in Supplementary Materials), the transitions of physical mixture, filament and formulations from 40 to 60 • C demonstrated that the amorphous drug existed and gradually became less in them. Still, the drug did not distribute molecularly homogenous in the formulations, which might have potential implications to the dissolution findings. Additionally, the Tg of pure Soluplus ® was at 78.4 • C, which was close to the previous report [40]. The Tg of Soluplus ® decreased successively in the order of physical mixture, filament and formulations, indicating the mixture was more homogeneous and the plasticization of PEG 400 was more effective.

Weight
The average weight of the 3D-printed gastric-floating formulations is shown in Table  1. The average weights of the cylinder-15 (373.5 ± 9.8 mg), capsule-0 (372.3 ± 17.5 mg) and hemisphere-0 (374.5 ± 18.8 mg) were close to each other (p > 0.05) and consistent with the model designed, which made it easier for evaluating the influence of external shapes on the floating and drug-release behavior of the formulations. For the capsule-or hemisphere-shaped formulations, those with lower infill percentage had smaller average weights. The average weights of capsule-0 and capsule-15 were 372.3 ± 17.5 mg and 404.5 ± 10.3 mg (p < 0.01), and the average weights of hemisohere-0 and hemisphere-15 were 374.5 ± 18.8 mg and 389.7 ± 10.1 mg (p < 0.05), respectively.
The relative standard deviation (RSD) of the formulation weight was calculated according to the standard deviation and average weight in Table 1. The data obtained were in the range of 2.62% to 5.02%. The RSD of the weight of 3D-printed formulations was supposed to reflect the printing precision and reproductivity in certain aspects [41]. For FDM 3D-printed formulations at average weight between 300 mg to 500 mg, the RSD of weight was reported from 0.31% to 8.17% [13,29,[42][43][44][45][46], and the number of formulations reported for determining the weight variation was from 3 to 10 [29,42,47]. In this study, the RSD of weight (2.62%~5.02%) and the number of formulations (n = 10) were both within the scope of literatures.

Hardness
The hardness of formulations is shown in Table 2. All formulations had sufficient hardness in the two testing directions (shown in Figure 2). The hardness of formulations was observingly affected by the tested direction, infill percentage and axial symmetry. Firstly, a decrease in hardness was observed more so in direction B than in direction A, especially for the cylinder-and hemisphere-shaped formulations. The result was supposed to be owing to the layer-by-layer structure of 3D-printing formulations, as the lateral force to layers in direction B broke the adhesion between layers more easily than vertical force to layers in direction A. Secondly, the formulations with higher infill percentage showed higher hardness, which could be found in the comparation of data between capsule-0 and capsule-15 or between hemisphere-0 and hemisphere-15 in both directions. The increase in infill percentage was considered to reinforce the mechanical strength of the

Weight
The average weight of the 3D-printed gastric-floating formulations is shown in Table 1. The average weights of the cylinder-15 (373.5 ± 9.8 mg), capsule-0 (372.3 ± 17.5 mg) and hemisphere-0 (374.5 ± 18.8 mg) were close to each other (p > 0.05) and consistent with the model designed, which made it easier for evaluating the influence of external shapes on the floating and drug-release behavior of the formulations. For the capsule-or hemisphere-shaped formulations, those with lower infill percentage had smaller average weights. The average weights of capsule-0 and capsule-15 were 372.3 ± 17.5 mg and 404.5 ± 10.3 mg (p < 0.01), and the average weights of hemisohere-0 and hemisphere-15 were 374.5 ± 18.8 mg and 389.7 ± 10.1 mg (p < 0.05), respectively.
The relative standard deviation (RSD) of the formulation weight was calculated according to the standard deviation and average weight in Table 1. The data obtained were in the range of 2.62% to 5.02%. The RSD of the weight of 3D-printed formulations was supposed to reflect the printing precision and reproductivity in certain aspects [41]. For FDM 3D-printed formulations at average weight between 300 mg to 500 mg, the RSD of weight was reported from 0.31% to 8.17% [13,29,[42][43][44][45][46], and the number of formulations reported for determining the weight variation was from 3 to 10 [29,42,47]. In this study, the RSD of weight (2.62~5.02%) and the number of formulations (n = 10) were both within the scope of literatures.

Hardness
The hardness of formulations is shown in Table 2. All formulations had sufficient hardness in the two testing directions (shown in Figure 2). The hardness of formulations was observingly affected by the tested direction, infill percentage and axial symmetry. Firstly, a decrease in hardness was observed more so in direction B than in direction A, especially for the cylinder-and hemisphere-shaped formulations. The result was supposed to be owing to the layer-by-layer structure of 3D-printing formulations, as the lateral force to layers in direction B broke the adhesion between layers more easily than vertical force to layers in direction A. Secondly, the formulations with higher infill percentage showed higher hardness, which could be found in the comparation of data between capsule-0 and capsule-15 or between hemisphere-0 and hemisphere-15 in both directions. The increase in infill percentage was considered to reinforce the mechanical strength of the formulations [48]. Thirdly, the symmetry of the formulation also affected its hardness behavior, especially when the force of the test direction was parallel to the layer. In the case of the cylinder-and capsule-shapes, the formulations were axial symmetric to the test force in both front and side view, and indeed exhibited good hardness. While the hardness of the hemisphere formulation significantly decreased where the shape was asymmetric in the side view. This phenomenon could be explained by the fact that the force added did not distribute evenly to the formulation, and broke it easily.

In Vitro Floating Behavior
The in vitro floating time of the 3D-printed formulations is shown in Table 2. There was no floating lag time observed in all the tests, as shown in Figure 9. The phenomenon was supposed owing to the density designed (0.638~0.827 g/mL, as shown in Table 1) which was lower than that of gastric juice (1.004 g/mL) [49]. The floating lag time was reported to raise the risk of gastric-floating formulations being expelled from the stomach before drug release [6]. Hence, the complicated work in traditional manufacturing processes, such as optimizing the formulation composition and the production process to avoid the floating lag time of gastric-floating formulations, was greatly reduced by the 3D-printing technology. On the other hand, as the infill percentage of the 3D-printed formulations was low in this study, the penetration of gastric fluid into the sealed center of formulation was easy, leading to the floating time being relatively short. A combinatory approach, such as a floating and expandable combinatory system, was suggested in a recent published literature which may help to overcome the limitation [50].

Drug Content
The average drug loading of the drug-loaded filaments was measured to be 9.90 ± 0.05% (w/w). Drug content of the formulations is shown in Table 2. The drug contents of drug-loaded filaments and printed formulations were between 99.1 to 102.2% of the drugs added in the preparation, indicating that there was no drug loss in either the HME or FDM 3D-printing processes.

In Vitro Drug Release
The in vitro drug release result is shown in Figure 10.  In this study, different external shapes of formulations were designed to investigate the influence of shape on the in vitro floating behavior. Though the theoretically calculated floating force of the hemisphere-or capsule-shaped formulations was larger than that of the cylinder-shaped formulations, the practical result revealed that the floating time of the hemisphere-and capsule-shaped formulations was shorter than that of the cylinder-shaped formulations (p < 0.05). During the in vitro floating test, the rupture of the hemisphereand capsule-shaped formulations was observed faster than that of the cylinder-shaped formulations, which was considered to be owing to the less connective area between filaments in the curved surface of hemisphere-and capsule-shaped formulations, compared with that in the flat surface of the cylinder-shaped formulations. Thus, it was easy for gastric fluid to penetrate into the hemisphere-and capsule-shaped formulations resulting in rapid expanding and the rupture of the formulation. Moreover, the rupture of the hemisphere-shaped formulations was found more quickly than that of the capsule-shaped formulations. The reason was assumed for the poorer axial symmetry of the hemisphere.
The floating time of capsule-0 and capsule-15 was 5.03 ± 0.03 h and 5.28 ± 0.06 h, and that of hemisphere-0 and hemisphere-15 was 4.24 ± 0.19 h and 4.55 ± 0.42 h, respectively. The result indicated that when comparing the same shape of the formulations, those with a higher infill percentage had a slightly longer floating time.

Drug Content
The average drug loading of the drug-loaded filaments was measured to be 9.90 ± 0.05% (w/w). Drug content of the formulations is shown in Table 2. The drug contents of drugloaded filaments and printed formulations were between 99.1 to 102.2% of the drugs added in the preparation, indicating that there was no drug loss in either the HME or FDM 3D-printing processes.

In Vitro Drug Release
The in vitro drug release result is shown in Figure 10. Compared with the commercial verapamil hydrochloride tablets, all the 3D-printed formulations achieved a similarly sustained release.

Conclusions
The drug-loaded HME filament composed of HPMC, Soluplus ® , PEG 400 and verapamil hydrochloride was newly developed and proved highly reliable for FDM 3D-printing sustained-release formulations. The gastric-floating cylinder-, capsule-and hemisphere-shaped formulations with low infill percentage (0% and 15%) were carefully designed and successfully fabricated through FDM 3D-printing technology. The 3D-printed gastric-floating formulations showed desired shapes, elegant appearance, suitable mechanical strength, and good constancy in both formulation weight and drug content. All the formulations floated successfully in vitro with no floating lag time, and released the drug sustainedly for more than 8 h. All the components were thermally stable throughout the whole preparation process and the verapamil hydrochloride was dispersed in an amorphous state, or molecularly. The formulations presented higher mechanical strength when the testing force was added vertically to the orientation of filaments or axial sym- The surface area of the three kinds of formulations (shown in Table 1) was designed close to each other in order to ensure a similar rate for water penetrating into the formulations. The results of drug release revealed that the formulations with higher weight released the drug more slowly. Capsule-15 was the heaviest formulation and showed the slowest drug release rate. The weight of cylinder-15, capsule-0 and hemisphere-0 was close, and their release profiles were almost overlapping (p > 0.05). In addition, the formulations with higher infill percentages showed slower release rates when comparing capsule-0 with capsule-15 or hemisphere-0 with hemsphere-15, which was consistent with the results reported in literatures [51,52].
As the 3D-printed formulations tended to stick on the paddles (in USP-II apparatus) and prolong the floating time in the drug release test, USP-I apparatus was used in this study [16]. To ensure the sufficient contact of formulations to medium, several dissolution methods were developed and reported [53]. Regarding the influence of in vivo gastric physiology (i.e., gastric motility, transit times, etc.) to the 3D-printed formulations, physiological parameters in the dissolution test, such as fluid volume, pH, agitation speed and so on, should be considered in the future work [54].

Conclusions
The drug-loaded HME filament composed of HPMC, Soluplus ® , PEG 400 and verapamil hydrochloride was newly developed and proved highly reliable for FDM 3D-printing sustained-release formulations. The gastric-floating cylinder-, capsule-and hemisphereshaped formulations with low infill percentage (0% and 15%) were carefully designed and successfully fabricated through FDM 3D-printing technology. The 3D-printed gastricfloating formulations showed desired shapes, elegant appearance, suitable mechanical strength, and good constancy in both formulation weight and drug content. All the formulations floated successfully in vitro with no floating lag time, and released the drug sustainedly for more than 8 h. All the components were thermally stable throughout the whole preparation process and the verapamil hydrochloride was dispersed in an amorphous state, or molecularly. The formulations presented higher mechanical strength when the testing force was added vertically to the orientation of filaments or axial symmetrically to the formulations. The floating time of the formulations depended on their ability to keep intact (not rupture), which was related to the external shape of the formulations. As the surface area of the formulations was designed to be similar, the drug release rate was mainly associated with the weight of the formulations. Moreover, this work demonstrated the potential of preparing FDM 3D-printed gastric floating beyond traditional formulation shapes and infill percentages, to control floating behavior and drug release.
Author Contributions: D.C., X.X., G.Y. and T.F. designed the experiment; D.C., R.L., H.Q. and X.X. performed the experiment; H.Q. and D.C. analyzed the data and wrote the draft preparation; T.F. revised and edited the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.