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Multi-Functional Self-Adhesive FiLM (Fibrous Layered Matrix) for Tissue Glues and Therapeutic Carriers Ye-Eun Yoon, Byung Gee Im, Jung-Suk Kim, and Jae-Hyung Jang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01413 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 12, 2016
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Multi-Functional Self-Adhesive FiLM (Fibrous Layered Matrix) for Tissue Glues and Therapeutic Carriers Ye-Eun Yoon, Byung Gee Im, Jung-suk Kim, and Jae-Hyung Jang*
Department of Chemical and Biomolecular Engineering, Yonsei University, 50, Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea
KEYWORDS Tissue adhesive, scaffolds, polycaprolatone, polyvinylpyrrolidone, dopamine hydrochloride, fibrous matrix
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ABSTRACT
Tissue adhesives, which inherently serve as wound sealants or as hemostatic agents, can be further augmented to acquire crucial functions as scaffolds, thereby accelerating wound healing or elevating the efficacy of tissue regeneration. Herein, multi-functional adherent fibrous matrices, acting as self-adhesive scaffolds capable of cell/gene delivery, were devised by coaxially electrospinning poly(caprolactone) (PCL) and poly(vinyl pyrrolidone) (PVP). Wrapping the building block PCL fibers with the adherent PVP layers formed film-like fibrous matrices that could rapidly adhere to wet biological surfaces, referred to as FiLM (Fibrous Layered Matrix) adhesives. The inclusion of ionic salts (i.e., dopamine hydrochloride) in the sheath layers generated spontaneously multi-layered fibrous adhesives, whose partial layers could be manually peeled off, termed d-FiLM (derivative FiLM). In the context of scaffolds/tissue adhesives, both FiLM and d-FiLM demonstrated almost identical characteristics (i.e., sticky, mechanical, and performances as cell/gene carriers). Importantly, the single FiLMprocess can yield multiple sets of d-FiLM by investing the same processing time, materials, and labor required to form a single conventional adhesive fibrous mat, thereby highlighting the economic aspects of the process. The FiLM/d-FiLM offer highly impacting contributions to many biomedical applications, especially in fields that require urgent aids (e.g., endoscopic surgeries, implantation in wet environments, severe wounds).
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INTRODUCTION Tissue adhesives, which are primarily employed as a hemostatic for bleeding controls or as a sealant for healing wounds, serve as key elements in many medical fields. In particular, in the context of regenerative medicine, tissue adhesives can further function as inter-mediators that can stably adhere biomaterial scaffolds to biological tissues 1-5 or as local depots that can act as a reserve for therapeutic agents (e.g., cells, growth factors, drugs, or gene vectors) to subsequently discharge factors at their implanted location to accelerate the achievement of the ultimate goals (e.g., tissue formation, wound cures)
6-10
. A variety of adhesive materials, including naturally
derived (e.g., fibrin glue, thrombin, or protein-based adhesives) and synthetic materials (e.g., cyanoacrylates, polyethylene glycol (PEG)- or urethane-based adhesives), have demonstrated favorable features for use in tissue adhesives: biologically inert and robust sticky properties 11, 12. Importantly, implementing advanced tissue adhesives, which can further accompany physical supports for carrying therapeutic agents (i.e., cells, drug/gene vectors), would contribute to numerous biomedical fields, including tissue engineering, surgical operations, endoscopic treatments, and cancer/gene therapy. The core capabilities that are essentially required for multi-functional, advanced tissue adhesives can be categorized into two purposes: i) for use as adhesive mediators, they must intimately interact and completely contact wet tissue surfaces that have irregular morphologies or uneven topographies 13 and ii) for use as local depots to accelerate wound healing processes, they must incorporate therapeutic agents (i.e., cells, drugs/genes) into their interiors for subsequent local releases at lesion sites 14. Therefore, the multi-functional tissue adhesives could potentially serve as i) sealants for wound closures, ii) interfaces for coupling therapeutic materials to local lesion sites, and iii) medicine for triggering tissue/wound repairs. To meet the first category as an adhesive mediator, the majority of existing adhesives have been designed as viscous gluey
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formulations, thereby lacking mechanically resistant capacities that can overcome harsh environments, such as high shear (e.g., blood streams), large incisions (e.g., severe wounds), or physically distorting conditions (e.g., dynamic motions)
15
. Thus, developing adhesive,
mechanically rigid substrates, which can also meet the second purpose as a physical support, is an alternative strategy for creating advanced tissue adhesives. However, current approaches to simultaneously achieve the two purposes generally require additional post-processes to glue finally generated physical substrates onto biological surfaces, such as suturing with adhesive agents
18, 19
16, 17
or coating
. Then, reciprocal interactions between the resulting substrates and the
adhesive agents must be further considered, or uniform spread of the adhesive agents throughout complicated networks of the substrates may work as a challenge. Furthermore, the long-lasting reaction times of adhesive agents for stably interlocking non-adhesive objects into wet surfaces may be challenging for their broad applications, for example, emergent surgeries (e.g., urgent blood control, organ transplantation) or endoscopic operations that typically require in situ rapid adherence of therapeutic agents. Alternatively, the development of a three-dimensional scaffold, which itself can rapidly exhibit adhesive features in wet environments, would be a facile strategy to create advanced tissue adhesives that fully satisfy the two goals mentioned above. In this study, highly versatile extracellular matrix (ECM)-like fibrous infrastructures that simultaneously possess the key principles of tissue adhesives and tissue engineering scaffolds without further processing (i.e., surface modification for adhesive properties) were developed. A building-block portion, poly(ε-caprolactone) (PCL; core), with an adhesive material, poly(vinyl pyrrolidone) (PVP; sheath), were successfully merged by coaxial-electrospinning of the dual materials (i.e., PCL@PVP matrices). The crosslinked PVP hydrogels have been representatively used as adhesives that can be readily adhered to wet biological tissues 20, 21. The inclusion of PCL
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fibers in the core along with the adhesive PVP parts in the exterior sheaths was necessary to overcome the PVP’s lack of mechanical strength. Most importantly, the proposed tissue adhesives were spontaneously composed of multiple fibrous layers, which were formed via saltinduced electrospinning
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. The multiple fibrous layers could be readily split into several
individual fibrous layers, which demonstrated almost identical characteristic properties (i.e., adhesive, morphological, and mechanical aspects) compared to the original whole adhesive fibrous blocks. Thus, by investing the same processing time, materials, and labor required to form single flat fibrous adhesive mats, many more thinner fibrous tissue adhesives could be obtained, confirming the economically efficient process. The original fibrous matrices with multiple layers were referred to FiLM (i.e., Fibrous Layered Matrix) adhesives, and the individually separated adhesives were referred to d-FiLM (i.e., derivative FiLM). The characteristic properties of both FiLM and d-FiLM were investigated, and their performances as tissue adhesives and scaffolds were examined by comparing with conventional non-adhesive PCL matrices, multi-layered PCL matrices without PVP, and single-layered PCL@PVP fibrous adhesives. In addition to their adhesive features, the FiLM/d-FiLM adhesives demonstrated versatile capabilities as cell or gene delivery vehicles, thereby fully presenting their potential as multi-functional tissue adhesives.
EXPERIMENTAL SECTION Fabrication of FiLM/d-FiLM adhesives Fibrous layered-matrix (FiLM) adhesives were fabricated by coaxially electrospinning poly(ε-caprolactone) (PCL, Mn = 80,000 g/mol: Sigma-Aldrich, St. Louis, MO, USA) and poly(vinyl pyrrolidone) (PVP, Mn = 360,000 g/mol: Sigma Aldrich), which was supplemented
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with an ionic salt, dopamine hydrochloride (dopamine; (2-(3,4-dihydroxyphenyl)ethylamine hydrochloride. Mn = 189.64 g/mol, Sigma, St. Louis, MO), as schematically depicted in Figure 1A. Briefly, 15% (w/v) PCL and PVP solutions were separately prepared by dissolving in a mixture of chloroform (99.0%, Duksan Pure Chemicals, Ansan, Korea) and N,Ndimethylformamide (DMF) (99.9%, Duksan Pure Chemicals) (v/v = 1:1) and a mixture of ethyl alcohol (99.9%, Duksan Pure Chemicals) and DMF (v/v = 1:1), respectively. Two additives, 0.2% (w/v) dopamine and 0.3% (w/v) 4,4’-diazidostilbene-2,2’-disulfonic acid disodium salt (DAS; Tokyo Kasei Kogyo, Tokyo, Japan), which were required to generate the multi-layered fibrous structures
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and to solidify the PVP parts under a UV source
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, respectively, were
included in the PVP solution prior to electrospinning. Each polymer solution was loaded separately into two syringes (23 G, 10 mL; NanoNC, Seoul, Korea) and coaxially ejected (PCL: core, PVP + dopamine (DA) + DAS: sheath) using electrospinning equipment (ESR100, NanoNC, Seoul, Korea) at a feed rate of 1.5 mL/h under high voltage (18 – 20 kV; working distance: 20 cm). The other PCL@PVP sets with different PVP portions were fabricated by varying the flow rates of the core and sheath layer: PCL@PVP (1:1) = 1.5 mL/h (PCL): 1.5 mL/h (PVP), PCL@PVP (1:2) = 1 mL/h : 2 mL/h, PCL@PVP (1:3) = 0.75 mL/h : 2.25 mL/h, and PCL@PVP (1:4) = 0.6 mL/h : 2.4 mL/h. Due to the low solubility of dopamine in the organic solvent (i.e., a mixture of chloroform and DMF), 0.2% ionic salt (i.e., dopamine monomer) was included in the sheath layer comprising PVP. In addition, the dopamine salts were included in the sheath layers to enhance the electrostatic interactions between the resulting fibers and to facilitate its removal from the final constructs upon the leaching process. Prior to the leaching process, the resulting matrices, which contained DAS as a photo-cure initiator, were exposed to UV radiation (230 nm; INNO-CURE 100N, Lichtzen, Ansan, Korea) to solidify the PVP sheath
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layer. These additives (i.e., dopamine salts (DA) and DAS) were removed by immersing in deionized water three times due to their detrimental effects on cellular viability. Three different control conditions were fabricated: i) conventional thin PCL fibrous mats (c-PCL), which were formed by electrospinning 15% (w/v) PCL solution dissolved in a mixture of chloroform and DMF (1:1), ii) non-adhesive multi-layered PCL matrices without PVP, which were dissolved in a mixture of chloroform and DMF (1:1) and electrospun with 0.2% dopamine, and iii) adhesive single-layered PCL fibrous matrices (no dopamine), which were formed by coaxially electrospinning 15% PCL (core) and 15% PVP/0.3% DAS (sheath) followed by photo-curing under UV light (i.e., single-layered PCL@PVP). All resulting fibers were kept in a desiccator (Dry Active, Korea Ace Scientific, Seoul, Korea) until use to remove residual solvent. The dFiLM, which originated from the FiLM blocks, was obtained by manually splitting the FiLM into several fibrous matrices using two sharp forceps. Each resulting separated matrix was referred to as a d-FiLM (derivative-FiLM).
Characterization of FiLM/d-FiLM adhesives The adhesive, physical, mechanical, and morphological features of the resulting fibrous structures were characterized. Initially, to confirm the core-shell structures of the FiLM, the fluorescence dyes coumarin (Sigma-Aldrich) and rhodamine B (Sigma-Aldrich) were included in each polymer solution (i.e., PCL and PVP, respectively) prior to fiber production, and the resulting matrices were observed under a confocal laser scanning microscope (CLSM, LSM700, Carl Zeiss, Thornwood, NY, USA) at the Yonsei Center for Research Facilities. The fibrous morphologies of the cross-sections or external surfaces of the resulting fibrous matrices were visualized using field emission scanning electron microscopy (FE-SEM) (JEOL-7001 F, JEOL
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Ltd., Tokyo, Japan) that was operated under 15 kV. The exterior SEM images were imported into ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the fiber diameters and porosity of each fibrous set were measured using the Orientation J plugin. The adhesive force of the resulting fibrous matrices on the small intestine obtained from the porcine peritoneal cavity, as a tissue example, was evaluated through a shear-adhesion test using a universal testing machine (Multi Test 1-i, Mecnnesin, Slinfold, UK). Briefly, a fragment of porcine intestine was placed along with each fibrous matrix (10 mm (width) × 10 mm (length)) at room temperature, and a 45 N pulling-force load was exerted on the fibrous matrices at a shear rate of 19 mm/min to measure the shear adhesion (N). The shear adhesion was subsequently normalized to the area of each fibrous matrix (i.e., N/cm2). Additionally, to further confirm the robust adhesive properties of the FiLM/d-FiLM systems, three additional adhesion tests were performed: i) an adherence test for which each fibrous matrix was adhered onto the surface of porcine intestine, and water-jets with a constant flow rate (1 mL/s) were exerted on the fibrous matrices; ii) a suturing test for which each fibrous matrix was adhered onto the cutting edges of porcine intestines that were completely cut into two pieces, and one end of the pieces was hung at a fixed point to test its suturing capability; and iii) a sealing test for which each fibrous matrix was used to block holes that were intentionally made using a punch, and a blueink stream with a 10 mL/s flow rate was passed through the intestine to check for leakages. All measurements were conducted in triplicate. The mechanical strength of each matrix was accessed by examining its stress-strain behaviors, which were measured using a universal testing machine (Multi Test 1-i, Mecnnesin, Slinfold, UK). The wettability of each fibrous set was measured using a contact angle meter (CAM 101, KSV Instruments Ltd., Espoo, Finland).
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Cell culture Four different cell types, NIH3T3 (mouse embryo fibroblast cell line), Jurkat (human T lymphocyte cell line), HEK293T (human embryonic kidney cells), and AAV293 cells, were utilized as model cells to verify the FiLM system as cell culture/delivery vehicles (NIH3T3 and Jurkat), gene delivery vehicles (HEK293T), and cell sources (AAV293) to package adenoassociated viral (AAV) vectors. The AAV293 cells are a HEK293T cell line genetically specialized for packaging AAV vectors (Stratagene, La Jolla, CA, USA). Three cell types, NIH3T3, HEK293T, and AAV293, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA), which was supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin (Life Technologies), and streptomycin (Life Technologies), at 37 °C and 5% CO2. The Jurkat cell lines were cultured in Roswell Park Memorial Institute medium (RPMI; Invitrogen, Carlsbad, CA, USA), which was supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin (Life Technologies), and streptomycin (Life Technologies), at 37 °C and 5% CO2
Analysis of cell viability and distribution within the fibrous matrices To determine whether the resulting fibrous sets possessed a favorable environment for growing cells, the metabolic activities of NIH3T3 cells grown within each matrix for 2 and 7 days were assessed using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) according to the manufacturer’s manual. Briefly, each fibrous set was sterilized by immersing in 70% ethanol prior to cell seeding, freeze-dried, and kept in a 48-well tissue culture plate (TCP). Subsequently, NIH3T3 cells (1×105 cells/matrix) were seeded on top of the matrix and grown on the 48-well TCPs for 2 or 7 days prior to
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analysis. At each time point (2 and 7 days post-culture), a 10% volume of CCK-8 solution relative to the culture medium was directly added to each well containing fibrous sets with cells, which were further incubated for 3 hours at 37 °C. Finally, the supernatants were collected, and their colorimetric changes at 440 nm were measured using a spectrophotometer (NanoDrop 2000, Thermo Scientific, West Palm Beach, FL, USA). The absorbance values at 440 nm were assessed to evaluate the viability of the cells grown within the fibrous sets. To visualize the cellular distribution across the fibrous matrices, the nuclei of NIH3T3 cells were stained with 4’6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 30 min, and the cellular distribution within each fibrous matrix was imaged using a CLSM. Finally, the distribution of fluorescencelabeled cells was reconstructed three-dimensionally using Imaris 7.4.2 software (Bitplane, Zurich, Switzerland).
In vivo analysis of the adherence of fibrous matrices and cellular infiltration In vivo performances of the FiLM/d-FiLM systems as tissue adhesives and tissue engineering scaffolds were evaluated by two sets of experiments: i) the adherence of fibrous matrices on the skin of mice and ii) subcutaneous implantation of the fibrous matrices. For the first test, each fibrous set was adhered to small incisions that were intentionally made on the back skin of female 8-week-old ICR mice (29-31 g; n=3), and the surrounding tissues with the fibrous matrices were retrieved at 20 minutes post-adherence. This test was performed to examine the rapid adhesion and the contact extents of all fibrous sets on biological tissues. The interfaces between the fibrous matrices and skins were primarily examined by staining cells at the proximity of the matrices using hematoxylin and eosin (H&E; Sigma-Aldrich). For the second test, each fibrous set was subcutaneously implanted into the backs of female 8-week-old
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ICR mice (29 - 31 g; n=3), and the resulting fibrous matrices with surrounding cells were retrieved at 2 and 7 days post-implantation. All of the experimental procedures followed the guidelines for the care and handling of laboratory animals and were approved by the Yonsei Laboratory Animal Research Center Institutional Animal Care and Use Committee (YLARCIACUC, Yonsei University, Korea). Cells within or adjacent to fibrous matrices were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C and subsequently immersed in 30% sucrose solution at 4 °C for 3 days. The tissue sections were frozen in optical cutting temperature compound (OCT) at -80 °C and cut into 50 µm thickness using a cryostat microtome (Leica, CM1850, Leica Biosystems, Wetzlar, Germany). The sections were then stained with H&E (SigmaAldrich), and the nuclei and cytoplasm of surrounding the cells were observed under a brightfield microscope (Nikon Eclipse Ti, Nikon Corporation, Tokyo, Japan).
AAV production and evaluation of fibrous matrices as gene delivery vehicles An AAV vector, AAV r3.45, which previously demonstrated enhanced gene delivery capabilities in various clinically valuable cell types, was used to evaluate the performances of the FiLM systems as gene delivery vehicles. Recombinant AAV r3.45 vectors carrying cDNA coding for green fluorescence protein (AAV-GFP) driven by a cytomegalovirus (CMV) promoter were packaged by transient transfection, as previously described
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. Briefly, three plasmids (17 µg
each), including a CMV GFP vector plasmid containing the inverted terminal repeat (ITR) (pAAV CMV GFP), an AAV helper plasmid (carrying cap r3.45), and an adenoviral helper plasmid (Stratagene), were complexed using calcium phosphate for efficient delivery to AAV293 cells. The resulting transfected cells were lysed to harvest the viral vectors, which were subsequently purified using a 1 mL HiTrap heparin column (GE Healthcare, Pittsburgh, PA)
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according to the manufacturer’s instructions. Finally, DNase-resistant genomic titers were determined by quantitative PCR (qPCR) (Mini Opticon, Bio-Rad, Hercules, CA). Finally, the amount of AAV immobilization and released from each fibrous set was quantified by measuring the intensity of fluorescence tagged on the AAV vectors. AAV vectors were fluorescently labeled using an Alexa Fluor 488 microscale protein labeling kit (Life Technologies), and the intensities of fluorescence collected in the supernatant after AAV immobilization or in the medium at designated time points (i.e., 6, 12, 24, 48, 72, 144, and 192 hours) were assessed. To quantify the cellular transduction, HEK293T cells (105 cells/matrix) were plated onto the fibrous matrices containing AAV vectors. At 2 days post-transduction, the cellular transduction within each fibrous set was evaluated by visualizing the localization of GFP-expressing cells across the fibrous matrices. At 2 days post-transduction, the localization of GFP-expressing cells across the fibrous matrices was imaged under a CLSM, and their distribution within fibrous matrices was three-dimensionally reconstructed using Imaris software. The nuclei of surrounding cells were counter-stained using DAPI.
Controlled Jurkat cell delivery from fibrous sets To assess the capability of the FiLM/d-FiLM systems to deliver cells, suspending Jurkat cells (5 × 106 cells/matrix) were seeded on each fibrous matrix (i.e., c-PCL, m-PCL, sPCL@PVP, FiLM, and d-FiLM), and the numbers of cells released from each matrix were manually counted at designated time points (i.e., 6, 12, and 36 hours) using a hemacytometer. Once cells were placed within each fibrous set, the resulting fibrous blocks were immersed in fresh media and transferred to a well containing fresh medium at each time point. Finally, cells that were collected at the medium at each time point were used to determine the cell delivery
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profiles. Because Jurkat cells are not adherent, cells could be readily collected at each time point without trypsinization.
Statistics All statistical analyses were performed using a one-way analysis of variance (ANOVA) with a post hoc Dunnett’s test using the SPSS 21.0 software package (IBM Corporation, Somers, NY, USA). P values
