Three-Dimensional Electrohydrodynamic Printing and Spinning of

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Applications of Polymer, Composite, and Coating Materials

3D Electrohydrodynamic printing and spinning of flexible composite structures for oral multi-drug forms Shuting Wu, Jing-Song Li, John Mai, and Ming-Wei Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08880 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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ACS Applied Materials & Interfaces

3D Electrohydrodynamic Printing and Spinning of Flexible Composite Structures for Oral Multi-Drug Forms Shuting Wu1,2, Jing-Song Li1, John Mai3, Ming-Wei Chang1,2* 1

Department of Biomedical Engineering, Key Laboratory of Ministry

of Education, Zhejiang University, Hangzhou 310027, P.R. China. 2

Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular

Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou 310027, P.R. China. 3

Alfred E. Mann Institute for Biomedical Engineering at the

University of Southern California, CA, US

Keywords: sandwich structure, drug delivery, multi-drug, 3D EHD printing, electrospinning

1

ACS Paragon Plus Environment

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Abstract A simple method to rapidly customize and to also mass produce oral dosage forms is arguably a current bottleneck in the development of modern personalized medicine. Specifically, delayed-release mechanisms with well-controlled dosage profiles for combinations of traditional Chinese herbal extracts and Western medications are not well established. Herein, we demonstrate a novel multi-drug loaded membrane sandwich with structures infused with ibuprofen (IBU) and Ganoderma lucidum polysaccharide (GLP) using 3D electrohydrodynamic (EHD) printing and electrospinning techniques. The resulting flexible membrane consists of micro-scaled, multi-layered cellulose acetate (CA) membranes loaded with IBU in either the shape of concentric squares or circles, as the top and bottom layer of a sandwich structure. In between the CA-IBU layers are randomly electrospun (ES) polyvinyl pyrrolidone (PVP) layers loaded with GLP. The complete fibrous membrane sandwich can be folded and embedded into a 0-size capsule to achieve oral compliance. Simulated in vitro testing of gastric and intestinal fluids demonstrated a triphasic release profile. There was an immediate release of GLP after gastric juices dissolved the capsule shell and the PVP, followed by the short-term release of 60% of the IBU within an hour afterwards, and the remaining IBU was sustained released following a Fickian diffusion profile. In summary, this multi-drug (both hydrophilic and/or hydrophobic) oral system with 2


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precision-designed

structures

should

enable

personalized

therapeutic dosing.

1. Introduction The emergence of personalized medicine to improve patient health care can arguably be traced to successful, low cost sequencing of the human genome.1 The genetic variation in individual patients can manifest in different responses to various drugs treatment therapies, and thus personalized medicine requires unique formulations and dosages for each patient in order to maximize the therapeutic benefits.2 Some advantages of personalized medicine include enhanced efficiency for target discovery, reduced costs for clinical trials, and fewer side effects for optimal outcomes.3 Broadly, personalized drug combinations tailored to each patient to achieve synergistic effects are becoming increasingly common in the pharmaceutical and healthcare industry.4 Drug combinations can simultaneously address side effects associated with traditional separate dosage medications, as well as improving patient compliance via convenient single dosing and reducing the risk of wrong or missing medicines, which will enable tangible multi-stage 3



improvements

in

healthcare.5

Despite

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several

successful

therapeutic outcomes,6-7 combining traditional herbs and western medicine into a single formulation is still a challenge. Oral unit doses are a commonly used route for drug administration due to their easy manufacturability, portability and convenience which promotes patient compliance.8 Hence, controllable drug delivery systems of oral forms (e.g. tablets, capsules, etc.) are an important motivating factor for improving therapeutic outcomes.9-11 Currently, oral tablets are usually fabricated by compression of drug-excipient materials because of their easy preparation.12-13 However, such mass manufacturing processes has limitations in personalized medicine due to the uncertain local concentrations and dose variations as the tablets dissolve.14 To address this challenge, many researchers have employed 3D printing technique to produce controllable caplets for personalized medicine. For example, 3D printing using hot melt extrusion (HME) and fused deposition demonstrate

modelling

(FDM)

customized

method

have

pharmaceuticals.15

been

used

However,

to

these

techniques may be not suitable for drugs with lower thermal

4


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stabilities, leading to drug degradation and loss of potency at the high processing temperatures.16 Additionally, the maintaining micro-scaled accuracies of these complex structures over large scale manufacturing production remains a huge challenge for customized 3D printing of oral drug carriers. This further limits the number of available materials (hydrophilic and hydrophobic doses) and the preparation methods that are compatible with current 3D printing technologies. Electrospinning is a cost-effective and versatile technique for fabricating fibers on the nanometer and micrometer scales from naturally occurring biomolecules to molten synthetic polymers for a wide range of applications.17-19 Electrohydrodynamic (EHD) printing is capable of patterning fibrous materials via digitallycontrolled deposition of materials (often layer by layer) to create well-ordered free form geometries, unlike the random structures obtained when using electrospinning.20-21 The spatial deformation of 3D printed structures for formulations in the gastrointestinal tract is drawing increasing attention due to the prolonged periods of time in the gastric cavity which may contribute to swelling or

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changes in elastic deformation of the synthesized materials.22-23 Generally, these drug delivery systems should be safe and nontoxic, and the dosage forms need to be small enough to take orally before deformation, while subsequently prolonging and improving drug release to ensure the safety and efficacy of such systems. Motivated by the aforementioned considerations, we have developed

a

flexible

membrane

sandwich

with

structures

fabricated by a combination of solution-based 3D EHD printing and electrospinning that can be loaded with multiple drugs and then folded

into

an

orally-administered

capsule.

After

capsule

dissolution, these flexible membrane sandwich remain deformable, thus maintaining longer continuous drug dosing in gastrointestinal tract. The membranes can be custom fabricated into different shapes for precision drug dosage which can be triggered from two separate membranes as the interlayer dissolves. Specially, the composite membrane sandwich is loaded with both Chinese herbs (Ganoderma lucidum polysaccharide, GLP) and Western medicine (ibuprofen, IBU). Both drugs can be independently released in a 6


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controllable manner for customized and personalized dosage forms.

2. Materials and methods 2.1. Materials Cellulose acetate (CA, Mw = 3l104 g/mol), polyvinyl pyrrolidone (PVP, Mw = 1.3 106 g/mol) and ibuprofen (IBU, ≥98%) were all purchased

from

Sigma-Aldrich,

USA.

Ganoderma

lucidum

polysaccharide (GLP) was obtained from TianHe Agricultural Group (Longquan, Zhejiang, China). Ethanol, acetone, N, NDimethylformamide (DMF) and phosphate buffer saline (PBS, pH= 7.4) were all supplied by Sinopharm Chemical Reagent, China. Deionized water (DI water) was produced in-house using a Millipore Milli-Q Reference ultra-pure water purifier, USA. Minimum Eagle’s medium (MEM, Gibco) and fetal bovine serum (FBS) were purchased from Invitrogen, USA. Penicillin and streptomycin were obtained from Biyuntian, China. Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto,

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Japan). All chemicals and reagents were analytic grade and utilized without further purification. 2.2. Preparation of EHD printing and electrospinning solutions CA powder was dissolved in a mixture of acetone / DMF (acetone:DMF=1:1, v/v) at a concentration of 22% (w/w) under mechanical stirring (VELP ARE heating magnetic stirrer, Italy) for 6 hours to ensure complete dissolution. IBU (5% w/w of CA) was then added to the CA solution and dissolved by mechanical stirring for 2 hours to obtain a homogenous IBU-loaded CA solution for EHD printing. PVP powder was dissolved in ethanol at a concentration of 13% (w/w) under mechanical stirring for 4 hours to obtain a PVP solution. GLP was added in DI water at a concentration of 2.5% (w/w) under stirring for 1 hour to prepare a GLP solution. Then, the GLP solution was added in the PVP solution at a volume ratio of 1:4 with constant stirring until homogenous mixing to obtain a GLP-loaded PVP solution for electrospinning. 2.3. Preparation of composite membrane sandwich

8


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Figure 1a, 1b and Figure S1 (Supporting Information. EHD and electrospinning setup.) show the main devices used in the fabrication process. The 3D EHD printing device consists of a stainless steel metallic nozzle (outer and inner diameters = 0.9 and 0.7 mm, respectively), a syringe pump (KD Scientific KDS100, USA), a high voltage power supply (Glassman high voltage Inc. series FC, USA) and a programmable X-Y-Z movement stage. The electrospinning device consists of a stainless steel metallic nozzle (outer and inner diameters = 0.9 and 0.7 mm, respectively), a syringe pump (KD Scientific KDS100, USA), a high voltage power supply (Glassman high voltage Inc. series FC, USA) and a collector. The 3D EHD printing set-up was used to prepare the concentric circular-shaped and square-shaped membranes, and the ES set-up was used to prepare the disordered fibrous layers. During the 3D EHD printing process, IBU-CA solution was loaded into and subsequently infused from a 5 ml syringe directly into the needle at a rate of 0.3 ml/h. A conductive glass substrate was mounted on to the X-Y-Z movement stage as the collector substrate, and the distance between the substrate and processing

9



nozzle tip was adjusted to 3 mm. The applied voltage delivered to the nozzle was set to 2 kV. The printing speeds (as the motion of deposition stage followed a pre-determined path) of concentric circular shape and concentric square shape were regulated as 21 and 22 mm/s, respectively. The precise movement of the X-Y-Z stage was programmed (ADT-TP3340, Adtech, China) by entering the X-Y-Z coordinates of each point into the control system, and by setting the number of desired repetitions (for Z axis control), the multi-layer 3D structure was fabricated, which enabled the deposition of up to 14 fibrous layers per membrane. The 3D printed membranes were then moved into the ES device and electrospun coated for 1 h. PVP-GLP solution was infused into the nozzle at a controlled rate of 0.85 ml/h using infusion pumps under the influence of an electric field (17.5 kV). The collector distance was set to 17 cm. The inset images in Figure 1b and Figure S1 (Supporting Information. EHD and ES setup.) show the semi-finished layers after ES coating for 10 min. Then, the two composite membranes were stacked into a sandwich structure as shown in Figure 1c. The final fabricated composite membrane 10


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sandwich was packed into an easy to swallow capsule. All these steps were carried out at the room temperature (25 ℃). 2.4. Fiber characterization The morphologies of the printed membranes with different geometric structures and randomly-oriented ES fibrous layer were investigated by scanning electron microscopy (SEM, FEI Quanta 650, Netherland). For imaging, the samples were sputter-coated with gold for 90 s with a ~50 nm thick gold coating and scanned at an accelerating voltage of 8 kV. The diameters of the printed CAIBU fibers were measured using ImageJ software (National Institute of Health, USA) from optical micrographs (OM，Phenix BMC503-ICCF, China). The mean diameter was determined by averaging 30 randomly selected fibers from each printed structure. The diameters of the ES PVP-GLP fibers were measured using ImageJ software (National Institute of Health, USA) from SEM micrographs. The mean diameter was determined by averaging 100 randomly selected fibers. The error bars were calculated from the standard deviation. All data were exported for analysis, and the

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distribution

graphs

were

plotted

using

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Original

software

(Originallab, USA). 2.5. Fourier transform infrared (FTIR) spectroscopy The FTIR spectra of CA, IBU, PVP, GLP and their formulations were analyzed using Fourier transform infrared spectroscopy (FTIR, IR Affinity 1, Shimadzu, Japan). The spectra were recorded between 400 and 4800 cm-1 at a resolution of 4 cm-1. 2.6. X-ray diffraction (XRD) To determine the physical form of each component (CA, IBU, PVP and GLP) in the composite membrane sandwich, the XRD patterns were analyzed using a X-ray crystal diffractometer (Gemini A Ohra, Oxford, UK). The samples were scanned over a 2 theta range of 5 ° to 60 ° with a step size of 0.02 ° at 40 kV and 40 mA. 2.7. Mechanical characterization The mechanical properties of the samples were evaluated by a universal material testing machine (Zwick/Roell Z020, Zwick, Germany).

Three

experiments

(14-layer

circular-shaped

membrane, 14-layer square-shaped membrane and the composite 12


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membrane sandwich) were performed before drug release. Each sample was loaded into the grips and then tested at a speed of 10mm/min until the sample ruptured. Then, after drug release after 0.5 and 24h, the membranes were analyzed by the same method in order to characterize any changes in their mechanical properties. Each experiment was performed in triplicate. 2.8. Membrane sandwich detachment behavior test The composite membrane sandwich detachment behavior was tested in vitro. Briefly, in order to observe the behavior of enteric capsulated composite membrane sandwich, the membrane sandwich-loaded capsule was placed into a simulated intestine model (HM733, HuaMao Group, China) and monitored by a camera (Baumer TXG02C, Germany). To observe the detachment behavior more clearly, the test was also performed by placing the composite membrane sandwich in a container with 800 ml of PBS at 37 ℃. The detachment behavior of the membrane was monitored by a camera again. 2.9. In vitro drug release

13



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The simulated gastric fluid (SGF, pH=1.7) and simulated intestinal fluid (SIF, pH=7.4) were prepared from a HCl solution and a phosphate buffered saline (PBS) to mimic their pH values,24-25 respectively. Each sample comprised of one piece of drug-loaded membrane with a known weight and 40 ml release medium. In order to simulate the route of oral administration, all assays were carried out in a HZ-8801K thermostatic oscillator (Taicang Science and Education Factory, China) at 37 ±0.5 °C in SGF for 2 hours first and then in SIF for 5 hours. As shown in Figure S2 (Supporting Information. UV absorption curves), the characteristic UV absorptions of IBU and GLP were determined to be 222 nm and 243, respectively (UV-2600 spectrophotometer, Shimadzu, Japan).

The

linear

standard

curves

were

obtained

from

standard solutions (Figure S3, Supporting Information. Standard dissolution curves). At regular intervals, 3 ml of the drug release solution was transferred for UV absorption spectra measurement, and then 3 ml of fresh medium was replenished into the release solution as the experiment continued. Each experiment was

14


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performed in triplicate. The cumulative release percentage of drug was calculated by Equation 1.19 Drug release % =

× 100%

(1) Here, Ct is the drug concentration in the solution at time t and CM is the maximum concentration of drug in the solution. 2.10. Kinetics of drug release Zero-order, first-order, Higuchi and Korsmayer–Peppas curvefitting models were applied to determine the drug release kinetics of IBU from the composite membranes in the simulated acidic gastric environment (pH=1.7) and neutral intestinal environment (pH=7.4).26-27 The model with the highest correlation coefficient (R2) was considered as the optimal fit. 2.11. In vitro cell culture and biocompatibility analysis For orally administered medications, low toxicity and good biocompatibility is required. To investigate the potential cytotoxic risk of the experimental products (CA-IBU membrane, PVP-GLP layer and their composition), L929 mouse fibroblast cells were 15



used to assess cytotoxicity in vitro cell culture. L929 cells were cultured in MEM medium with 10% FBS and 1% antibioticantimycotic solution in a cell culture dish at 37 °C, in 5% CO2. The culture medium was changed every 2 days. The printed CA-IBU membrane was added into the culture medium at a concentration of 1 mg/ml. The ES PVP-GLP was added into the culture medium at a concentration of 1 mg/ml. All samples were sterilized under UV light for 5 h before experiment. 100 µL of a uniform L929 cell suspension was added to a 96-well plate and incubated for 24 h. CA-IBU, PVP-GLP and their mixture were added to the cell culture plate. A Cell Counting Kit-8 (CCK-8) assay was used to evaluate proliferation of L929 cells at designated time intervals (4 and 8 h) during cell culture. 20 µL CCK-8 solution were added to each well and then incubating for further 3 h at 37℃, 450 nm absorbance was recorded using a microplate reader (Multiskan GO, Thermo Fisher Scientific, USA). Cell viability in a plate with no samples and the culture medium with CCK-8 solution were used as the control and the blank, respectively. The relative cell viability is presented as Eq (2).28 16


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Cell viability % =

. !" #. !$% . &'$()'! #. !$%

× 100 %

(2) Where, Ab.(sample) means the absorbance of sample groups, Ab.(control) means the absorbance of the control group and Ab.(blank) means the absorbance of blank group. In addition, for cell biocompatibility, L929 cells were seeded on the composite membranes. After 2 days incubation, cells were fixed by 4 (v/v) % formalin and permeabilized by 0.1% Triton X-100. Alexa Fluor 546 phalloidin (Invitrogen, California, USA) and 4′, 6′diamidino-2-phenylindole hydrochloride (DAPI, Invitrogen) were used to stain the cell cytoskeleton and nuclei, respectively. The cell distribution on the membranes was visualized using a fluorescent microscope (Nikon Ti-S, Japan). 2.12. Statistical analysis All experiments were performed in triplicate for each sample. Data is presented as mean ± standard deviation. Statistical analysis was performed using SPSS software (SPSS Statistics v20, IBM, U.K.) by Student’s t-test. Differences are considered statistically

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significant when *p

Three-Dimensional Electrohydrodynamic Printing and Spinning of

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