A 3D-printed local drug delivery patch for pancreatic cancer growth suppression

Journal of Controlled Release 238 (2016) 231–241

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A 3D-printed local drug delivery patch for pancreatic cancer growth suppression Hee-Gyeong Yi a,1, Yeong-Jin Choi b,1, Kyung Shin Kang c, Jung Min Hong c, Ruby Gupta Pati a, Moon Nyeo Park a, In Kyong Shim d, Chan Mi Lee d, Song Cheol Kim d,e,⁎, Dong-Woo Cho a,⁎⁎ a

Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk, Republic of Korea Division of Integrative Biosciences and Biotechnology, POSTECH, Pohang, Kyungbuk, Republic of Korea Department of Anatomy & Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA d Biomedical Engineering Research Center, Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea e Department of Surgery, University of Ulsan Colleague of Medicine & Asan Medical Center, Seoul, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 11 November 2015 Received in revised form 16 April 2016 Accepted 8 June 2016 Available online 8 June 2016 Keywords: 3D printing Local drug delivery Biodegradable patch Pancreatic cancer

a b s t r a c t Since recurrence and metastasis of pancreatic cancer has a worse prognosis, chemotherapy has been typically performed to attack the remained malignant cells after resection. However, it is difficult to achieve the therapeutic concentration at the tumor site with systemic chemotherapy. Numerous local drug delivery systems have been studied to overcome the shortcomings of systemic delivery. However, because most systems involve dissolution of the drug within the carrier, the concentration of the drug is limited to the saturation solubility, and consequently cannot reach the sufficient drug dose. Therefore, we hypothesized that 3D printing of a biodegradable patch incorporated with a high drug concentration would provide a versatile shape to be administered at the exact tumor site as well as an appropriate therapeutic drug concentration with a controlled release. Here, we introduce the 3D-printed patches composed of a blend of poly(lactide-co-glycolide), polycaprolactone, and 5-fluorouracil for delivering the anti-cancer drug in a prolonged controlled manner and therapeutic dose. 3D printing technology can manipulate the geometry of the patch and the drug release kinetics. The patches were flexible, and released the drug over four weeks, and thereby suppressed growth of the subcutaneous pancreatic cancer xenografts in mice with minimized side effects. Our approach reveals that 3D printing of bioabsorbable implants containing anti-cancer drugs could be a powerful method for an effective local delivery of chemotherapeutic agents to treatment of cancers. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Pancreatic cancer is a highly aggressive disease because of its tendency for early local spreading and metastasis. Chemotherapy is often used after resection of the cancer to prevent recurrent growth and metastasis of remaining malignant cells. Nevertheless, a low survival rate in the range, in the range of 8–25%, has been reported following extended Abbreviations: 5-FU, 5-flurouracil; PLGA, poly(lactide-co-glycolide); PCL, polycaprolactone; MHDS, multi-head deposition system; SEM, scanning electron microscope; FT-IR, Fourier transform infrared spectroscopy; PBS, phosphate buffered saline; DMSO, dimethyl sulfoxide; MTS, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)tetrazolium, inner salt; IC50, inhibitory concentration 50; hASC, human adipose stem cell; H&E, hematoxylin and eosin; i.v., intravenous; s.d., standard deviation; S:V, surface area to volume. ⁎ Correspondence to: S. C. Kim, Department of Surgery, University of Ulsan College of Medicine & Asan Medical Center, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul 05505, Republic of Korea. ⁎⁎ Correspondence to: D.-W. Cho, Department of Mechanical Engineering and Center for Rapid Prototyping-based 3D Tissue/Organ Printing, POSTECH, 77 Cheongam-ro, Nam-gu, Pohang, Kyungbuk 37673, Republic of Korea. E-mail addresses: [email protected] (S.C. Kim), [email protected] (D.-W. Cho). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.jconrel.2016.06.015 0168-3659/© 2016 Elsevier B.V. All rights reserved.

surgery [1,2]. Since most chemotherapeutic drugs, such as 5-flurouracil (5-FU), paclitaxel, and cisplatin, have poor aqueous solubility, conventional administration techniques (e.g., intravenous injection, oral administration) cannot easily deliver a therapeutic concentration of the drugs at the tumor site. In addition, anti-cancer agents may accumulate in crucial organs (e.g., heart, liver, lung) and cause severe side effects [3, 4]. Therefore, various local drug delivery systems have been developed to overcome the shortcomings of conventional chemotherapy. The major objectives of local drug delivery systems are to enhance drug dosage, to improve overall quality of life, and to minimize systemic side effects. Injectable or implantable systems display spatiotemporal controllability and prolonged release kinetics. A biodegradable triblock copolymer poly(lactide-co-glycolide) (PLGA)-polyethylene glycolpoly(lactide-co-glycolide) was developed as an injectable hydrogel containing the dissolved paclitaxel; it exhibited a fairly long release period (6 weeks) [5]. A poly(ester-carbonate)-collagen composite film releasing hydroxycamptothecin was found to be flexible and could be fixed onto tissue; it also prevented the new growth of lung cancer [6]. However, chemical dissolution of drugs into delivery systems is limited by the saturation solubility of the drug in the carrying material [7].

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Conversely, if the drug is physically dispersed as a powder inside the carrying material, the final concentration can be above the saturation solubility. Intratumoral injection of PLGA microspheres containing a high content of 5-fluorouracil (5-FU) crystalline powder significantly decreased mortality in glioma-bearing rats [8]. Blending of 5-FU or paclitaxel powder into ethylene-vinyl acetate film demonstrated the effective local exposure at a high drug concentration for a long-term release period [9]. Currently, three-dimensional (3D) printing technology is driving major innovations in the biomedical field, including drug delivery. 3Dprinted drug-incorporating constructs can be designed and fabricated with precisely defined architecture and dose. 3D printing allows the drug to be released from a construct in a controlled manner by manipulating the geometry of the construct. Among the various kinds of 3D printing systems, the extrusion-based method provides flexibility in selection of drugs and polymers, wide range of concentration of the incorporated drugs, solvent-free continuous dispensing of the drugcontaining material, and uniform distribution of the drug [10]. The antibiotic-loaded polycaprolactone (PCL) constructs fabricated using the extrusion-based printing techniques proved that the drug delivery systems could be produced to have high loading efficiency and good stability of the incorporated drug, even if the drug was subjected to high temperatures during the manufacturing process [11,12]. We hypothesize that a 3D-printed biodegradable patch containing a high concentration of chemotherapeutic drug could be easily applied to the exact tumor site and reduce the cancer growth. For this study, we blended PLGA and PCL (PLGA/PCL), loaded with different amounts of 5-FU into the blend, and printed PLGA/PCL/5-FU patches. We assessed whether the 3D printing process is suitable for producing a highly concentrated drug delivery system. The printed patches were evaluated in vitro to identify their characteristics including mechanical properties, release kinetics, and therapeutic feasibility. We then analyzed the therapeutic efficacy and systemic toxicity by applying the patches to a mouse model of pancreatic cancer. We demonstrate that the 3D-printed PLGA/PCL patches can deliver 5-FU for more than four weeks and thereby suppress the cancer growth, with reduced side effects. 2. Materials and methods 2.1. 3D printing of PLGA/PCL/5-FU patches 5-FU and PLGA (lactide:glycolide = 85:15) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and PCL was purchased from Polysciences, Inc. (Warrington, PA, USA). 5-FU crystalline powder was comminuted using a mortar and a pestle, and then sieved through a 100-μm nylon mesh to ensure extrusion through a nozzle with a 250μm inner diameter. After melting the PCL and PLGA granules together at a ratio of 1:1 and 140 °C for 5 min on a glass dish, the 5-FU powder was then manually mixed into the molten polymers at 140 °C for 5 min using a thin rod (Fig. 1A). The PLGA/PCL/5-FU paste was loaded in the printing head of inhouse extrusion-based 3D printer called a multi-head deposition system (MHDS) [13]. The MHDS is composed of a nozzle-connected printing head (metal syringe and heating block), pneumatic pressure controller, and three-axis (X, Y, Z Cartesian coordinates) linear motion controllers. The PLGA/PCL/5-FU was pneumatically extruded from the reservoir at 600 kPa and 140 °C and deposited on the stage along the designed path at room temperature (Fig. 1B). PLGA/PCL patches were loaded with no drug, 10 mg, 50 mg, 100 mg, and 150 mg of 5-FU per gram of total weight (P0, P10, P50, P100, and P150, respectively). We fabricated various P150 patches with different pore shapes and total thicknesses to evaluate the effects of patch geometry on the release profile. While the layers were stacked, we altered the orientation of the lines in each layer to generate various pore shapes (Fig. 1C). The angles between the lines of subsequent layers were

designed as follows: 90° between the lower and upper layers to generate a latticed pattern; 90° between the lower and sandwich layers, and 60° between the sandwich and upper layers to generate a slant pattern; and 60° between the lower and upper layers to generate a triangular pattern. The lattice-type patches with 2, 4, and 8 layers (2L, 4L, and 8L, respectively) were fabricated to increase the total thickness of the patches. For use in in vitro and in vivo experiments, the circular lattice patch was selected to demonstrate feasibility. We expected that the lattice pattern has a low Young's modulus compared to the other patterns, as demonstrated in our previous study [14]. The circular shape was designed to cover the rounded shape of the solid tumor in the in vivo experiment. The latticed pattern was designed to have the same width gap and strut, so the vertical and horizontal lengths of the pore were equal. 2.2. Analysis of physical characteristics of 3D-printed patches We used a scanning electron microscope (SEM; Hitachi SU-6600, Hitachi, Tokyo, Japan) to observe the surface morphologies of the 3Dprinted patches. The samples were platinum-coated by a sputter-coater and imaged using an accelerating voltage of 15 kV. A tensile test was performed to identify the tensile properties of the printed patches. Test specimens were prepared following ISO 527-1 standards. Rectangular specimens (10 mm × 50 mm × 0.2 mm) with latticed pores were fabricated. We prepared the specimens made of only PLGA, only PCL, PLGA/PCL, and PLGA/PCL/5-FU (150 mg/g of specimen) for comparative study. A single-column Instron 3340 mechanical testing system (Instron, Norwood, MA, USA) was utilized. The specimens were gripped 10 mm from each end and stretched at a strain rate of 0.027 s−1 (n = 4 per each group, Fig. S1). 2.3. Analysis of chemical characteristics of 3D-printed patches The practical amount of 5-FU in the patches was determined. A weighted patch was dissolved in a mixture of 1 ml of dichloromethane and 5 ml of distilled water. The polymeric part and 5-FU were dissolved in the dichloromethane and the water, respectively [15,16]. The emulsion was centrifuged to separate the phases. The concentration of 5FU dissolved in the water of the upper phase was determined by measuring the absorbance at a wavelength of 265 nm using a UV–Vis spectrophotometer (Agilent 8453, Agilent Technologies, Palo Alto, CA, USA). We assessed the quality of 5-FU in the printed patches by conducting Fourier transform infrared spectroscopy (FT-IR). Potassium bromide discs containing 0.5% w/w of pure 5-FU, small pieces of PLGA, PCL, and the 3D-printed PLGA/PCL/5-FU patch were prepared separately. The sample spectra were obtained using an FT-IR spectrometer (n = 3 per each group; IFS-55, Bruker, Billerica, MA, USA). 2.4. 5-FU release, swelling, and degradation study The release kinetics were obtained by soaking the printed patches in 1 ml of phosphate-buffered saline (PBS) at 37 °C (n = 5 per each group) [11,17]. All released media were harvested and replaced with fresh PBS at regular intervals during the release periods. The concentration of 5FU in the release media was determined by measuring the absorbance at λ = 265 nm using the UV–Vis spectrophotometer. We observed the swelling and degradation of the patches to determine the mechanism of 5-FU release. After accurately determining the initial weight (WI), the patches were separately incubated in 5 ml of PBS at 37 °C. We harvested, immediately measured the swollen weight (WS), and then calculated the degree of swelling as follows (n = 5 per each group): Degreeofswelling ½% ¼ 100ðW S −W I Þ=W I For the degradation study, the incubated samples were collected for 4 months. The samples were freeze-dried, then the dry weights (WD)

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Fig. 1. 3D printing of 5-FU-loaded patch. (A) Process of blending PLGA, PCL, and 5-FU. (B) Schematic drawing describing the system apparatus of MHDS, an extrusion-based 3D printer. (C) CAD of three types of pores latticed, slant, and triangular types.

were measured. To quantify the degradation of patches, we calculated the residual weight as follows: Residualweight ½% ¼ 100W D =W I

2.5. Evaluations of chemosensitivity and therapeutic effects in vitro MIA PaCa-2 (ATCC, Manassas, VA, USA), a human pancreatic cell line, was used to determine chemosensitivity to 5-FU as previously described [17]. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% v/v fetal bovine serum (Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL, Grand Island, NY, USA) at 37 °C in 5% CO2 in a humidified incubator. The culture medium was refreshed every 2 to 3 days. After harvesting cells, 2 × 104 cells were plated on each well of a 24-well culture plate and incubated for 24 h prior to drug treatment (n = 5). The cells were then exposed to various concentrations of 5-FU from 10−2 μg/ml to 102 μg/ml for 24 h. The 5-FU was serially diluted in dimethyl sulfoxide (DMSO) and added to the culture medium at a ratio of 1:100. As a control, the same volume of drug-free DMSO was dissolved in the culture medium. After treatment, the medium was gently aspirated, and the cells were washed with fresh DMEM and incubated in drug-free culture medium for 72 h. We determined relative cell viability by performing 5(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4sulfophenyl)tetrazolium, inner salt (MTS) assay using a commercial MTS solution (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA), then calculated the median inhibitory concentration (IC50).

We then evaluated the cytotoxic effect of the printed PLGA/PCL/5-FU patches on malignant and non-malignant cells. As a model of non-malignant cells, we selected human adipose-derived stem cells (hASCs). In order to allow the concentration of the released 5-FU from P10 patch in culture media to be above the IC50-value for 24 h, the required volume of the media was calculated based on the release profile and set to 200 μl. P0, P10, P50, P100, and P150 patches were each immersed in the 200 μl of culture media, in which MIA PaCa-2 cells or adipose stem cells had been plated, separately (n = 5 per group). 2.6. Generation of pancreatic cancer model and implantation of the 3Dprinted patches To assess the therapeutic effect of the 3D-printed PLGA/PCL/5-FU patches in vivo, we implanted the patches to the bottom of subcutaneously grafted pancreatic cancers in athymic mice. The animal study was performed according to the protocol approved by the Animal Care and Use Committee of POSTECH (2014-01-0002). After 1 week to allow the mice to acclimatize to housing conditions, 1 × 107 MIA PaCa-2 cells in 100 μl of PBS were inoculated subcutaneously into the right flank region of 6-week-old male BALB/C nude mouse. After 3 days to allow formation of a tumor mass (average size = 266.5 ± 58.0 mm3), mice were implanted with P100 and P150 patches (n = 10 per group). The mice that received no treatment or were implanted with the P0 patch were also prepared for the comparison (n = 10 per group). We anesthetized mice by intraperitoneal injection of 250 mg/ kg 2,2,2-tribromoethanol (Sigma-Aldrich), made an incision near the inoculation site, inserted the patch right under the solid tumor, and immobilized the patch by suturing. All surgical procedures were performed in a specific-pathogen free room.

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2.7. Evaluations of therapeutic effects in vivo To estimate the tumor growth, we measured length (l, mm) and width (w, mm) 2 to 3 days using calipers, then calculated the tumor size as follows:   Tumorsize mm3 ¼ π  ðw=2Þ2  l The tumors were excised at 1 week and 4 weeks, fixed with 10% formalin, embedded in paraffin, and then sectioned. Tissue sections were stained with hematoxylin and eosin (H&E) to observe the cell morphology. Immunofluorescence staining was performed to examine proliferation of human pancreatic cancer cells in the tumor. We applied rabbit polyclonal Ki-67 anti-body (Abcam, Cambridge, MA, USA, 1:100) and mouse monoclonal human lamin antibody (h-lamin, Abcam, 1:100) overnight at 4 °C. The samples were then incubated with secondary antibodies including anti-rabbit IgG conjugated with FITC (Molecular Probes, Eugene, OR, USA, 1:200) or anti-mouse IgG conjugated with TRITC (Molecular Probes, 1:200), and DAPI for 1 h at 37 °C. Images were obtained using a Fluo View 1000 confocal microscope (Olympus, Melville, NY, USA) at constant camera settings. 2.8. Evaluations of systemic toxicity To visualize the drug release in vivo, we incorporated rhodamine B (Sigma), a well-known optical-imaging agent [18], instead of 5-FU, into the PLGA/PCL patches during the 3D-printing process. The rhodamine B-loaded patches were also prepared with the same dosages: 100 and 150 mg per g of the patch (denoted as P100′ and P150′, respectively). The mice received the implantation of P100′ and P150′ patches (n = 10 per group). After 1 and 4 weeks later, whole-body imaging was performed using the IVIS Kinetic Imaging System (Caliper Life Sciences, Hopkinton, MA, USA). The images were obtained with the excitation and emission wavelengths of 570 and 620 nm, respectively. After observing the external part of the whole-body, the internal part was examined by dissection. For the comparison, mice were intravenously (i.v.) injected with rhodamine B through the tail vein at a concentration of 60 mg/kg of mice weight, and observed under the same imaging settings for 6 days. Blood analysis was conducted to compare the systemic toxicity between the 5-FU injection (60 mg/kg of mice weight, once in every week; [19,20]) and the implantation of P100 and P150 patches. Plasma was isolated from heparinized blood samples at 0, 1, and 4 weeks after the implantation. 5-FU concentrations were measured using reversephase high-performance liquid chromatography with MS/MS detection (RHPLC-MS) by Korea Basic Science Institute (Daegu, Korea). To assess the liver and kidney functions, glutamic oxaloacetic transaminase (GOT) and blood urea nitrogen (BUN) were determined according to the routine procedure by Korea Research Institute of Bioscience and Biotechnology (Cheongwon, Chungbuk, Korea). 2.9. Statistical analysis We presented the data from the several experiments as means ± standard deviation (s.d.). When we compared more than two groups, we performed analysis of variance (ANOVA) for the statistical comparison. If the F-value in the ANOVA test rejected the null hypothesis, we conducted Bonferroni post-hoc test. Differences were regarded as significant at P b 0.05. 3. Results 3.1. Physical characteristics of 3D-printed PLGA/PCL/5-FU patches To demonstrate the versatility of the 3D-printing method, we fabricated the patches in square form without loops (20 mm × 20 mm), and

circular and oval forms with a loop on each side (8 mm in diameter for circular form, 8 mm in major length and 4 mm in minor length for oval form) to allow suturing (Fig. 2A). The 3D-printer also fabricated the various pore shapes with lattice, slant, and triangular patterns; the latticed patches were layered 2, 4, and 8 times (Fig. 2B, C). Thus, 3D-printing technology enables a drug delivery system with the diverse designs having the exact shape and size of the target region. Incorporation of 5-FU in the PLGA/PCL blended polymer influenced the surface morphology of the printed patch. The surface of the printed patches became rougher with the increase of the 5-FU content (Fig. 2D). The flexibility of the printed patches was also affected by incorporating 5-FU into PLGA/PCL blend. While PLGA showed brittle behavior, the stress-strain curve of the PLGA/PCL showed a much smaller slope, indicating increased flexibility (Fig. 2E). The PLGA/PCL incorporated 5-FU showed a slightly bigger slope than that of PLGA/PCL without drug. Nevertheless, the tensile modulus of PLGA was significantly highest than those of PCL, PLGA/PCL, or PLGA/PCL/5-FU (Fig. 2F). Thus, the ductility of PCL greatly contributed to increase flexibility of the 5-FU incorporated patch. As shown in Fig. 2G, the P150 patch is flexible and stretchable. Thus, it is suitable for covering a curved surface of soft tissue. 3.2. Chemical characteristics of 3D-printed PLGA/PCL/5-FU patches The theoretical amount of 5-FU in each patch was calculated from the concentration of 5-FU loaded and weight of patch. We then compared the theoretical amounts with the measured practical amounts. The average ratio of the practical amount of 5-FU to the theoretical amount of 5-FU (P/T ratio) was 101.2% (s.d. = 1.1) (Table 1). Thus, the process of blending 5-FU in PLGA/PCL and printing patches exhibited almost ideal efficiency for loading 5-FU in patches in the tested range, from 10 mg/g to 150 mg/g. The effect of the fabrication process on 5-FU should be examined because the blended material is subjected to temperatures up to 140 °C during the printing. FT-IR spectra of PLGA and PCL exhibit both absorption bands of the \\C\\H and C_O functional groups (PLGA: 1996 cm− 1 and 1733 cm− 1; PCL: 3042 cm− 1 and 1736 cm− 1) (Fig. 3). The spectrum of 5-FU shows the absorption band of the N\\H functional group (3424 cm−1). The spectrum of the printed PLGA/PCL/5-FU patch shows all absorption bands of N\\H,\\C\\H, and C_O functional groups (3426 cm−1, 3068 cm−1, 1722 cm−1, respectively). The data indicate that the loaded and printed 5-FU was not damaged by the temperature. 3.3. 5-FU release, swelling, and degradation study The various structures of P150 patches influenced the 5-FU release during the observation period (6 days). When the pore types were different, both slanted and triangular patches released much lower levels of 5-FU compared to the lattice patch (Fig. 4A). Besides, the increased total thicknesses of 4L and 8L patches significantly reduced 5-FU release. Thus, we assume that the drug release was affected by the changes of surface area depending on the geometric modifications. The tendency of reduction in drug release corresponds to the decrease of surface area to volume ratio (S:V ratio) in the tested structures, indeed (Table. 2). Therefore, the change of the structure using a 3D printing can easily alter the drug-release behavior. In the analysis of 5-FU release profile, each of the 3D-printed PLGA/ PCL/5-FU patches showed burst release in the first few hours, decreased release for 1 week, and then sustained release thereafter (Fig. 4A, Fig. S2). The 5-FU release was proportional to the amount of drug loaded in the patches. P150 released approximately 30% of the loaded drug over the 4-week period. To explore the release mechanism of 5-FU from the printed patches, swelling and degradation studies were performed. All patches swelled rapidly in the first hour, but the volume reached an asymptote within

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Fig. 2. 3D-printed P150 patches with various structures. (A) Three shapes of the patches: square without loops; circle and oval shapes with loops on each side for suturing. Scale bar: 5 mm. (B) Three types of pores: latticed, slanted, and triangular. (C) Lattice patches layered 2, 4, and 8 times (denoted as 2, 4, and 8L, respectively) Scale bar: 2 mm. (D) SEM images of surface of P0, P10, P50, P100, and P150 patches. Scale bar: 500 μm. (E) Stress-strain curves and (F) tensile moduli of PLGA, PCL, PCL/PLGA with or without 5-FU. *: P b 0.05 difference versus PLGA/PCL/ 5-FU (G) Photographs presenting the flexible and stretchable properties of P150 lattice patch. Scale bar: 2 mm.

24 h (Fig. 4B). The degree of swelling decreased as the amount of 5-FU increased. This trend may occur because 5-FU is hydrophobic. The P0 patch degraded very slowly: WD decreased by 29% over 120 days. The combination of increased hydrophobicity due to the loaded 5-FU, and slow degradation of the P0 patch suggests that degradation of the patches containing 5-FU should be slow. However, the weight loss of the patches increased with the loading amount of 5-FU (Fig. 4C). We assume that this phenomenon was due to the increased net loss, including both loss of polymers and 5-FU.

treatment groups decreased slightly at low concentration of 5-FU, but decreased steeply at concentrations N 1 μg/ml. The IC50 was 55.90 μg/ml. We then examined the cytotoxic and therapeutic effects of the 3Dprinted patches on the hASCs and the MIA PaCa-2 cells. We estimated the concentrations of released 5-FU from the patches to be above the IC50–value in 200 μl of culture medium (Table S1). All kinds of the PLGA/PCL/5-FU patches inhibited the proliferation of MIA PaCa-2 cells

3.4. Therapeutic effects in vitro MIA-PaCa-2 cells exposed to the serially diluted 5-FU died in a dosedependent manner (Fig. 5A). The relative cell viabilities of 5-FU Table 1 Mean ± s.d. (n = 4) theoretical and practical amounts of 5-FU in each patch. Patch

WI [mg]

Theoretical [μg]

Practical [μg]

P/T ratio [%]

P10 P50 P100 P150

4.5 ± 0.3 3.1 ± 0.1 3.1 ± 0.2 4.3 ± 0.1

44.8 ± 3.1 155.0 ± 7.1 310.0 ± 17.3 640.0 ± 17.3

49.1 ± 1.5 158.0 ± 3.1 295.1 ± 3.5 637.4 ± 15.2

105.4 ± 3.8 100.0 ± 3.3 98.4 ± 1.2 101.2 ± 2.4

Theoretical: weight proportion 5-FU × WI; practical: measured amount of 5-FU; P/T: Practical/theoretical.

Fig. 3. Infrared spectra of pure PLGA, pure PCL, pure 5-FU, and the printed PCL/PLGA/5-FU patch.

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Fig. 4. Release, swelling, and degradation profiles of 3D-printed PLGA/PCL/5-FU patches. (A) Cumulative 5-FU release (Cum. Rel.) profile of latticed, slanted, triangular patches and 4- and 8-layered lattice patches for 6 days. (B) Cum. Rel. profile of P10, P50, P100, and P150 patches for 32 days. The amount of 5-FU released was normalized with the initial weight. (C) Degrees of swelling and (D) residual weight profiles of the patches for 96 h and 4 months, respectively.

to less than 50% (Fig. 5B). From the hASCs, although the significant decreases in the cell viability were observed from the P100 and P150 patches, the overall cell viability was maintained N50% in all kinds of patches (Fig. 5B). Thus, the 5-FU dosages loaded in the patches were effective on the pancreatic cancer cell rather than on the non-malignant cell. 3.5. Therapeutic effects in vivo The release study conducted for 4 weeks indicated that the concentration of 5-FU released decreased after the burst release and equilibrated after 1 week. Thus, the inhibitory effect of 5-FU released from the patches should be examined for the same period as the release study. As we aimed to deliver the highly concentrated drug to cancer sites using the patches, we selected P100 and P150 patches for in vivo study. A previous study that applied a high dose of 5-FU (132 mg) showed effective treatment of cancer without the tissue damage in vivo [21]. Thus, we assumed that the survival of the non-malignant cells would be different in in vivo conditions, although the P100 and Table 2 Theoretical S:V ratio of the various geometries of P150 patches. Geometry

Lattice, 2L

Slant, 3L

Triangular, 3L

Lattice, 4L

Lattice, 8L

S:V ratio

22.20

20.30

20.29

9.64

8.91

2, 3, 4, and 8L: layered 2, 3, 4, and 8 times, respectively.

P150 patches showed some cytotoxicity on the non-malignant cells (56% and 62% of cell viability, respectively) in vitro. The patch was attached under the tumor (Fig. 6A). Tumor size significantly decreased in mice that received P100 or P150 patches compared to the P0 patch group (Fig. 6B). The size of the excised tumors from the mice implanted with P100 and P150 patches were much smaller than those of no treatment and P0 patch groups at four weeks (Fig. 6C). Interestingly, the tumor growth was inhibited more significantly in the mice implanted with P100 patch than in the mice implanted with P150 patch at 24 and 28 days (Fig. 6B). P150 decreased the tumor size more than the P100 did in the first 5 days, but thereafter the decrease in size was insignificant, whereas P100 decreased the tumor size constantly. We stained tissue sections of excised tumors to evaluate the effect of the released drug on cell morphology and proliferation in the tumors. In H&E histology, the tissue sections of both no treatment and P0 patchimplanted groups show the typical pattern of demarcated, densely-cellular neoplasm, and central hypoxia at 1 week (Fig. 7A). In immunofluorescence images (Fig. 7B), the human pancreatic cancer cells were detected by staining with h-lamin. The h-lamin-positive cells and hlamin-negative cells were also distinguishable in the two groups at 1 week, showing demarcated tumor formation. Ki-67 is a nuclear protein that is associated with cellular proliferation. The tumors of the no treatment and P0 patch-implanted groups exhibited large numbers of Ki-67-positive nuclei in both the tumor and the surrounding normal tissues. The tumors developed their invasive morphology in H&E and immunofluorescence images at 4 weeks. In

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Fig. 5. Therapeutic and cytotoxic effects of the 3D-printed patches. (A) Relative cell viability (Rel. cell viability) of MIA-PaCa-2 cells against the serial dilution of 5-FU. (B) Rel. cell viabilities of MIA-PaCa-2 cells and hASCs against the released 5-FU from P10, P50, P100, and P150 for 24 h.

H&E histology, hematoxylin-unstained nuclei indicate apoptotic cells were detected near the implanted patch in both P100 and P150 groups. The area of apoptotic cells was less extensive in the P100 group then that in the P150 group at 1 week. The P100 group also showed a narrow region of Ki-67-negative and h-lamin-positive cells near the patch. However, the tendency of P100 versus P150 on the apoptotic cells was reversed at 4 weeks. The P150 group showed strong staining of the nuclei in the tumor near the patch and showed damaged tissue of the skin on top of the tumor in H&E staining. Relatively more Ki-67-positive nuclei were observed in the tumors of the P150 group than in those of the P100 group. In contrast, the tumor mass on top of the patch was weakened and the extent over which Ki-67-positive nuclei of h-laminnegative cells was observed in the P100 group; these results suggest that damage to the surrounding normal tissue was minimized. As we observed from the in vitro results (Fig. 5B), the P150 patch has the higher toxicity on the non-malignant cells, compared to the P100 patch has, which may induce the tissue damage on the skin on top of the tumor. 3.6. Evaluations of systemic toxicity The rhodamine B administrated to the mice was visualized using in vivo imaging analysis. The injected rhodamine B spread over the whole-body right after the injection was shown with the highest

Fig. 6. Therapeutic effects of 3D-printed PLGA/PCL/5-FU patches on pancreatic cancer. (A) Photographs of the subcutaneous pancreatic cancer xenograft (T) before and after implantation of the patch (black arrowheads) at the bottom of the tumor. Scale bar: 1 cm. (B) Changes of relative tumor size of mice that received P100 and P150 patches against that of the P0 patch implantation group. *: P b 0.05 between P100 and P150 groups. (C) Photographs of the excised tumors and the patches at four weeks after implantation. The arrow indicates the flexibility of the implanted P150 patch. Scale bar: 2 mm.

fluorescent intensity at 30 min, and rapidly diminished for 6 days (Fig. 8A and C). On the other hand, the rhodamine B released from the 3Dprinted patches was detected only in the patch implanted region and with the similar intensity levels at 1 and 4 weeks (Fig. 8B, D). The released rhodamine B was not shown on the internal organs except for the P150′ at 1 week. The drug concentration in plasma was determined after administrating 5-FU into the mice through the i.v. injection, and implantation of P100 and P150 patches. The high level of 5-FU was detected in each group at the initial day of the implantation surgery (Fig. 8E). However, the 5-FU level in plasma significantly decreased in the patch-implanted mice at 1 and 4 weeks, except for i.v. group. The high level of 5-FU also influenced the liver function. While the mice in i.v. group showed constantly abnormal level of GOT for 4 weeks, the mice implanted with P100 and P150 patches showed similar levels to that of the P0 patch-implanted mice from 1 week (Fig. 8F). There was no significant change in the kidney function (Fig. 8G). The weights of the tumor-bearing mice

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Fig. 7. Morphology and proliferation in tumors at 1 and 4 weeks after the implantation of the 3D-printed patches. (A) H&E staining of tissue sections. Scale bar: 250 μm. (B) Immunofluorescence staining against Ki67 in green and human lamin (h-lamin) in red. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. T: tumor. P: implanted patch. Red arrows: non-proliferating tumor cells near the implanted patch. Green arrows: proliferating non-malignant cells near the implanted patch.

increased in all groups; this observation indicates that the mice could tolerate highly concentrated 5-FU released from the patches and suffer no obvious toxicity. Besides, no mice died in any group during the experiment period. Therefore, taken together, we demonstrated that the 3D-printed patch facilitates highly-localized drug delivery and minimization of systemic toxicity. 4. Discussion Here, we are exploring 3D-printed patches for the delivery of an anti-cancer drugs to specific regions near tumors. 3D printing technology provides a facile and versatile way to fabricate drug delivery platforms [22]. The diverse shapes of the printed patches suggest that extrusion printing would facilitate design effectively tailored to cover the margin of the diseased area. Additionally, as shown in Fig. 4A, the 3D printing process to control the pore shapes and total thickness of the patch allows specific modification of the release characteristics by altering the S:V ratio, which affects the diffusion rate. Goyanes et al.

also reported that the various 3D geometries influenced the drug release due to the alterations of S:V ratio [23]. Whereas the porosity of microspheres is determined by adjusting the use of solvents or emulsion process [24,25], 3D printing technology allows the direct manipulation of the geometry according to a specific purpose [26,27]. Currently, Particle Replication in Nonwetting Templates (PRINT) technology can produce the anti-cancer drug loaded particles by layer-by-layer process and can control the release profile by changing the particle's geometry or size or chemical composition as in 3D printing technology [28]. PLGA/PCL blend was chosen for our approach, because it accrues both advantages of PLGA and PCL. Although the chemical and mechanical characteristics of PLGA are tunable by altering the lactide:glycolide ratio [29], PLGA alone cannot achieve both a long drug release time and flexibility simultaneously. This is because the increased lactide proportion not only prolongs the release and degradation time frame, but also enhances the rigidity of the material. Therefore, the PLGA/PCL blended polymer has been widely studied for biomedical applications.

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Fig. 8. Analysis of the drug release in vivo. Time-dependent visualization of rhodamine B administrated by (A) the i.v. injection and (B) 3D-printed patch implantation. Time-dependent changes of fluorescence intensities in (C) the i.v. injection and (D) the P100′ and P150 patch implantation groups. (E) 5-FU concentration (5-FU Conc.), and (F) GOT and (G) BUN levels in the plasma harvested at the initial day, 1 week, and 4 weeks. (H) Changes in body weight of tumor-bearing mice with no treatment, P0, 100, and 150 patches. *: P b 0.05 vs. i.v. group. #: P b 0.05 vs. P0 group.

Cha et al. demonstrated the remarkably prolonged release and degradation time from the PLGA/PCL blend [30]. The inherent ductility of PCL could allow the 3D-printed PLGA/PCL/5-FU patch to be flexible and deformable to attach on the contours of soft tissue. 5-FU was used as a prototype drug in the present study because it has been broadly used in gastrointestinal cancers, head and neck cancers, and central nervous system cancers [21,31]. In particular, the

combination of 5-FU with high intensity focused ultrasound (HIFU) has been reported to enhance the efficiency of the drug delivery [32]. 5-FU has also been studied for use in the local drug delivery systems, but the investigation of therapeutic efficacy of these systems has yet to be exhibited [21,33]. Therefore, additional efforts are encouraged to explore advanced drug delivery systems that enable localized therapeutic effects.

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To establish proof-of-concept for delivering an anticancer-drug via a 3D-printed patch, we generated a pancreatic cancer model. Only less than 20% patients are candidates for surgical resection, because pancreatic cancers are usually detected at late stage when the cancer has already metastasized to other organs [34]. Moreover, among the patients received surgical resection, the residual cancer cells easily cause the local recurrence [35]. Thus, implantation of a local drug delivery system during the surgery would be ideal to suppress the remnant microscopic tumor, and to inhibit the tumor recurrence. Indeed, for the treatment of brain tumor, Gliadel Wafer®, a carmustine incorporating biodegradable wafer, has been utilized to implant in the resection cavity following surgical removal. Similarly, we expect that the 3Dprinted patch can also be directly implanted onto the tumor-removed region. In particular, 3D printing technology provides many advantages to generate local drug delivery systems. Because this technology allows to build a construct as desired, the 3D-printed patch can be fabricated with a specific shape that is fit to the tumor-resected site; thereby, it may benefit palliative effect as well as improving the quality of life for some patients suffering from late-stage pancreatic cancer [3]. Furthermore, the 3D-printed patch can be also applied to other types of cancer including prostate, colon, uterine corpus, and female breast cancer, which are diagnosed at early locoregional stage. Despite surgical resection, these cancers leave undetected microscopic cancer cells, which lead to cancer recurrence. Thus, the 3-printed patch can be also be effective for local therapy for these kinds of cancer. The important requisites for designing this application are the prolonged and sustained release of the drug, and administration of a dosage within the therapeutic window without inducing systemic toxicity [3]. In this study, the printed PLGA/PCL patches released approximately 20% of 5-FU over 4 weeks and degraded over 4 months. Although a further release study is required to track the total loss of the drug, the P100 and P150 patches showed therapeutic effects during the experiment period. Interestingly, the administered concentration of 5-FU by attaching a single P100 patch (180 mg/kg of mouse weight) is approximately 3 times that of intravenous bolus injection (60 mg/kg), while the mice showed no obvious side effects such as decreased liver function and weight loss. The 3D-printed PLGA/PCL/5-FU patch have potential as an effective vehicle for delivering drugs to achieve both high efficacy and minimized systemic toxicity for cancer treatment by enabling precise control of the amount and location of drug loaded.

5. Conclusions We developed a biodegradable patch that contains a high concentration of an anti-cancer drug for a localized administration of the drug to the exact site of a tumor. 3D printing of the porous patches with the diverse shapes demonstrated the ability to manipulate the drug release by altering the surface area. Besides, the PLGA/PCL/5FU patches exhibited the attachable flexibility, prolonged release period, and a significant suppressive effect on the growth of a subcutaneous pancreatic cancer xenograft with minimized side effects. These results indicate that use of 3D-printed patches will allow effectively localized delivery of anti-cancer drugs while achieving an ideal pharmacokinetics.

Acknowledgment This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2010-0018294) and by a grant from the Korean Health Technology R & D Project, Ministry of Health & Welfare, Republic of Korea (grant no. HI14C2640).

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2016.06.015.

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