Plasma ion implantation of silk biomaterials enabling direct covalent

This configuration leads to difficulties in processing objects that do not have ... mesh, placed 5 cm in front of the sample holder and electrically c...
1 downloads 4 Views 3MB Size
Subscriber access provided by Kaohsiung Medical University

Biological and Medical Applications of Materials and Interfaces

Plasma ion implantation of silk biomaterials enabling direct covalent immobilization of bioactive agents for enhanced cellular responses Alexey Kondyurin, Kieran Lau, Fengying Tang, Behnam Akhavan, Wojciech Chrzanowski, Megan S. Lord, Jelena Rnjak-Kovacina, and Marcela M.M. Bilek ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03182 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Plasma ion implantation of silk biomaterials enabling direct covalent immobilization of bioactive agents for enhanced cellular responses Alexey Kondyurin1&, Kieran Lau2, Fengying Tang2, Behnam Akhavan1,3, Wojciech Chrzanowski4,5, Megan S. Lord2, Jelena Rnjak-Kovacina2 & Marcela Bilek*1,3,5,6 1

School of Physics, University of Sydney, Sydney, NSW 2006, Australia Graduate School of Biomedical Engineering, UNSW Sydney, Sydney, NSW 2052, Australia 3 School of Aerospace, Mechanical and Mechatronic Engineering, University of Sydney, NSW 2006, Australia 4 Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia 5 Sydney Nanoscience Institute, University of Sydney, NSW 2006, Australia 6 Charles Perkins Centre, University of Sydney, NSW 2006, Australia 2

* Corresponding Author

& present address is Farm at Ewingar, Ewingar, NSW 2469, Australia

Abstract Silk fibroin isolated from Bombyx mori cocoons is a promising material for a range of biomedical applications, but it has no inherent cell-interactive domains, necessitating functionalization with bioactive molecules. Here we demonstrate significantly enhanced cell interactions with silk fibroin biomaterials in the absence of biofunctionalization following surface modification using plasma immersion ion implantation (PIII). Further, PIII treated silk fibroin biomaterials supported direct covalent immobilization of proteins on the material surface in the absence of chemical cross-linkers. Surface analysis after nitrogen plasma and PIII treatment at 20kV, revealed that the silk macromolecules are significantly fragmented and, at the higher fluences of implanted ions, surface carbonization was observed to depths corresponding to that of the ion penetration. Consistent with the activity of radicals created in the treated surface layer, oxidation was observed on contact with atmospheric oxygen and the PIII treated surfaces were capable of direct covalent immobilization of bioactive macromolecules. Changes in thickness, amide and nitrile groups, refractive index and extinction coefficient in the wavelength range 400-1000 nm as a function of ion fluence are presented. Reactions responsible for the restructuring of the silk surface under ion beam treatment that facilitate covalent binding of proteins and a significant improvement in cell interactions on the modified surface are proposed. Keywords Silk, biomaterial, plasma immersion ion implantation, covalent biofunctionalization, cell interactions

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

TOC Graphic:

1. Introduction Silks are protein-based, fiber-forming materials spun by a range of living organisms, including silkworms, spiders, and many other arthropods. While historically used in the textiles industry and as a medical suture, the last two decades have seen an extraordinary increase in the use of Bombyx mori silk fibroin (referred to as ‘silk’ throughout) materials for applications including tissue engineering, regenerative medicine, biomaterials, drug delivery, and optoelectronic applications.1-3 This progress in utility is underpinned by improved understanding of the structure-function relationship of silk and advances in silk fiber processing into reconstituted silk, an aqueous silk solution that serves as the source material for various material formats engineered from silk.2 Silk can be engineered into thin films, fibers, porous scaffolds, micro- and nano-particles, 3D printed structures, and hydrogels.2 The amino acid sequence of silk contains glycine-alanine-glycine-alanine-glycine-serine (GAGAGS) repeats that self-assemble into an anti-parallel beta-sheet structure. These beta-sheets are crystalline and crosslink silk through strong intra- and inter-molecular hydrogen bonds and Van der Waals interactions giving silk its robust mechanical properties.4 While B. mori silk is easily processed into a range of biomaterial formats and possesses excellent mechanical and degradation properties, it has no inherent cell interactive domains and its hydrophobic nature limits cellular interactions.4-5 To achieve improved biological function, silk biomaterials have been modified using a range of techniques including passive adsorption, covalent cross-linking, and bulk loading with bioactive molecules.1, 6 Biofunctionalization of silk biomaterials is required for a range of applications, including development of silk-based corneal7, vascular8, orthopedic9-10 and wound healing constructs11-12, as well as modulation of non-specific protein interactions with silk biomaterials13. Passive adsorption can result in a random distribution of molecular conformations, displacement by serum proteins and thus compromised biological performance.14 Covalent coupling is a more stable modification compared to adsorption and bulk loading, and the resultant interface is often more uniform. However, silk has a very limited number of modifiable amino acid side chain groups4, making physical surface modifications attractive. There is extensive literature reporting changes in the surface chemistry, structure, and properties of silk after physical surface modifications including UV, gamma, electron

ACS Paragon Plus Environment

2

Page 3 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

beam, and plasma treatment. Typical observations report fragmentation and crosslinking of the silk macromolecules followed by reactions with atmospheric species such as oxygen.15-16 However, most of the literature concerns modification of silk fibers and textiles. A limited number of studies have been devoted to understanding structural transformations in B.mori silk biomaterials fabricated from reconstituted silk and only a few of those have explored the biological efficacy of the physically modified silk biomaterials.17-21 In one study, electron beam treatment of silk and chitosan-coated bovine pericardium resulted in a moderate improvement in endothelial cell adhesion.22 Plasma treatment of B.mori silk biomaterials, including films, electrospun nanofibers and porous scaffolds, has been shown to improve cell adhesion and antithrombogenic properties relative to untreated silk. 17-19, 21 In one study, plasma treatment in methane was found to decrease the hydrophilicity of silk biomaterials, while plasma treatment in oxygen increased the hydrophilicity and enhanced interactions with human epidermal keratinocytes and dermal fibroblasts over a seven day period.19 Air plasma treated silk scaffolds were found to have an increased number of oxygen containing functional groups and increased wettability.21 When implanted in vivo in rabbit critical sized femur defects, the plasma treated silk scaffolds showed an enhanced osteoinductive effect compared to untreated scaffolds and accelerated rate of trabecular bone formation and defect healing.21 While there is evidence that plasma treatment of silk can result in enhanced protein immobilization using chemical cross-linkers, no study to date has demonstrated direct covalent immobilization of proteins on silk biomaterials. Oxygen and tetrafluoromethane-based plasma treatment of silk fabrics resulted in improved alkaline phosphatase enzyme activity when immobilized using cyanogen bromide chemical coupling.23 Nitrogen plasma was used to prepare the surface of silk films for free-radical polymerization with acrylic acid, thus increasing the number of carboxylic acid functional groups for further chemical modifications.24 These functional groups have been used as chemical handles to modify the surface chemistry of silk biomaterials to tailor their biological properties.24 Ion implantation is a process in which positively charged ions from plasma are accelerated and implanted into the surface of a material. Typically, the ions are accelerated to energies that are much higher than the bond energies between atoms in the materials being modified resulting in the breaking of bonds in the modified surface layer .25-26 The higher the energy of the implanting ions the deeper is the surface modification, typically extending tens of nanometers below the surface. Ion bombardment applied to polymers, results in substantial dehydrogenation and densification of the carbonaceous surface layer.25-27 The highly non-equilibrium restructuring process results in the formation of a predominately amorphous subsurface layer containing buried radicals. Relatively recently, it has been shown that these radicals can defuse to the surface to facilitate direct covalent immobilization of biologically active molecules providing an opportunity for simple, one-step surface bio-functionalization.28-29 Radicals and their formation of oxygencontaining groups upon reactions with atmospheric oxygen also make the surface mildly hydrophilic and therefore compatible with cells. 26, 28 As both the development of silk biomaterials and the use of plasma ion implantation for direct covalent functionalisation of the surfaces of polymers are relatively new advances, there are no

ACS Paragon Plus Environment

3

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 33

published attempts using plasma ion implantation of silk to achieve direct covalent bio-functionalisation to date. Ion implantation surface treatments have been used to enhance non-specific cellsurface interactions, including cell adhesion, proliferation and total surface colonization on the surfaces of a wide range of polymers.30 Ion implantation treatment is less well characterized on natural polymers than it is for synthetic polymers. For example, ion beam implantation decreased endothelial cell adhesion on collagen.31 Despite the increasing body of work on various surface treatments for silk materials, there are few studies of the effects of ion implantation on silk biomaterials for biomedical applications. Plasma immersion ion implantation (PIII) has significant practical advantages compared to traditional beam line ion implantation methods. 32 In the case of ion beam treatment processes, ions are extracted and accelerated away from remote plasma sources by large potential differences applied across meshes at the plasma source. This configuration leads to difficulties in processing objects that do not have simple 2D geometries. In contrast, in the PIII process, the object to be treated is immersed directly in the plasma and ions are accelerated to its surface by applying pulses of negative bias to the object or to a mesh placed around the object. These attributes make the PIII process particularly well suited to the surface modification of biomedical implants that typically have complex 3D geometries, such as those for vascular, wound healing and orthopedic applications. Any plasma chamber can be easily converted to a PIII system by adding a relatively low-cost high voltage pulsed power supply. In the present study, silk films were modified by PIII for enhanced cell interactions and direct protein immobilization, demonstrating a simple approach that can be utilized to biologically functionalize silk biomaterial surfaces for a broad range of biomedical device applications. To our knowledge, this is the first comprehensive report of the structural changes that occur in silk biomaterials fabricated from reconstituted silk during PIII treatment and the first demonstration of direct covalent protein immobilization on silk biomaterials in the absence of chemical cross-linkers.

ACS Paragon Plus Environment

4

Page 5 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

2. Materials and Methods 2.1. Reconstituted silk solution preparation Bombyx mori silk cocoons (Tajima Shoji Co., Ltd., Yokohama, Japan) were degummed by cutting and boiling 5 g of cocoons in 2 L of 0.02 M Na2CO3 (Sigma Aldrich) solution for 30 min to remove sericin. The washed and dried fibers were dissolved in 9.3 M lithium bromide (Sigma Aldrich) solution at 25 % wt/v for 3 h at 60°C. Silk solution was dialyzed against MilliQ water using 3.5 MWCO dialysis tubing (SnakeSkinTM, Life Technologies) for three days to remove lithium bromide from the solution. Remaining silk solution was centrifuged twice at 8700 rpm for 15 min at 4°C to remove debris. Silk solution concentration was determined by weighing a known amount of solution pre and post drying overnight at 60°C. Silk solution was stored at 4°C until further use. 2.2. Silk film preparation Silk films of 60 and 90 nm nominal thickness were prepared by spin coating the silk solution (2% wt/v, filtered through a 0.22 µm polyethersulfone membrane filter) onto silicon substrates (P-type, , 104 - 2×104 ohm.cm, one side polished, 0.610 0.640 mm thickness, Topsil, USA) at 2000 rpm using a SCS G3P-8 Spincoater. Silk solution concentration and spin coating rate were selected to give a homogenous coating thickness over the entire silicon wafer. The uniformity was confirmed by observing a uniform blue color distribution over the wafer. Silk films used for surface plasmon resonance and cell studies were prepared as described in Sections 2.9 and 2.10. 2.3. Plasma immersion ion implantation (PIII) treatment of silk films An inductively coupled radio-frequency (13.56 MHz) plasma, powered at 100 W with a reverse power of 12 W when matched, was used as the source of ions for PIII. 26 The base pressure of the vacuum chamber was 10-5 Torr and the pressure of nitrogen gas during the implantation process was 2·10-3 Torr. Silicon substrates coated with silk were mounted on a metal sample holder. Ion implantation occurred through a metallic mesh, placed 5 cm in front of the sample holder and electrically connected to it. Acceleration of ions from the plasma was achieved by the application of 20 kV bias pulses of 20 µs duration to the sample holder and mesh at a frequency of 50 Hz. To assess the effects of the plasma treatment in the absence of the accelerated ion impacts, the samples were placed on the same electrode but the bias was not applied. In this case, the sample holder was electrically floating. The plasma parameters were measured using a Langmuir probe (Hiden Analytical Ltd). The Langmuir probe was positioned over the substrate holder and consisted of a 0.20 mm diameter tungsten wire embedded in a sintered alumina ceramic tube inserted into a stainless steel tube grounded to the chamber. Software from Hiden Analytical Ltd. was used for data acquisition and analysis. The floating potential of the substrate holder was negative 40 - 45 V during plasma treatment and the plasma density was 2.6×109 ions/cm3. Ion fluence estimates for PIII treatment were obtained using the results of previous experiments on polyethylene 33-35 by a procedure described elsewhere in detail. 26 Briefly, the fluence of one high voltage pulse was determined by comparing UV transmission spectra from polyethylene films implanted under the conditions used here to samples implanted with known ion fluences under equivalent conditions in ion

ACS Paragon Plus Environment

5

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 33

beam treatment experiments where the fluence was measured with a Faraday cup. One second of PIII treatment under the present conditions was found to correlate with a fluence of 1.25 × 1013 ions/cm2. The silk samples were treated for durations of 40 – 800 s, corresponding to implantation ion fluences of 5 × 1014 – 1016 ions/cm2. The SRIM-2008 code 36 was used to predict the depth of nitrogen implantation into the silk coatings and the associated distribution of defects.

2.4. Spectroscopic ellipsometry The thicknesses and refractive index (n) of the silk coatings were determined using a Woollam M2000V spectroscopic ellipsometer before and after exposure to the plasma and PIII treatment. Ellipsometric data were collected for three angles of incidence65°, 70°, and 75°. In the case of the untreated coating, a model consisting of a transparent Cauchy layer on top of the silicon substrate was sufficient to achieve a good fit to the data. When fitting the data collected for PIII treated samples, a model with a transparent Cauchy layer was attempted initially. If a good fit was not obtained for all three angles of incidence, then an absorption factor was added to the Cauchy layer in the model. 2.5. Fourier-transform infrared (FT-IR) spectroscopy Transmission FTIR spectra were recorded using a Bruker Vertex 70v FTIR spectrometer before and after plasma and PIII treatments. The spectral resolution was 4 cm-1, and the number of scans was set to 500 to achieve a sufficient signal/noise ratio. Difference spectra between those taken from the silicon wafers with and without the silk coating were calculated and analyzed. The optical density of spectral lines associated with particular bond vibrations were used to quantify structural changes in the silk coating. 2.6. Silk coating stability tests Crosslinking was investigated by dissolving treated and untreated silk coatings in water for 10 minutes. Water is considered a “good” solvent for regenerated silk, as under laboratory conditions, silk films cast from regenerated silk completely dissolved in water. After exposure to water, the coatings were dried with compressed air and analyzed using ellipsometry and transmission FTIR. 2.7. Atomic force microscopy (AFM) The topography of untreated and PIII-treated silk before and after exposure to water was imaged using AFM (Park System) in tapping mode. An oscillation amplitude of 10 nm and a frequency of 1 Hz were applied for scanning the samples. The data were analyzed using Gwyddion software 37. The Root Mean Square (RMS) parameter was used to quantify the roughness of the surface topography. 2.8. Contact angle measurements and surface energy calculations The wettability of the silk samples before and after PIII treatment was measured using the sessile drop method with a Kruss DS10 contact angle goniometry. De-ionised water (polar liquid) and diiodomethane (non-polar liquid) were used as test fluids to measure the static contact angles. Surface energy and its polar and dispersive components were calculated using the Owens, Wendt, Rabel, and Kaelble model by a regression method. 2.9. Surface plasmon resonance (SPR) spectroscopy

ACS Paragon Plus Environment

6

Page 7 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

SPR spectroscopy was used to evaluate the protein binding on untreated and PIIItreated silk films. Silk solution (0.5% wt/v in deionized water, 300 µl), filtered through a 0.22 µm filter was spin-coated onto BIAcore gold chips at 300 rpm for 6 s followed by 2000 rpm for 60 s. The silk-coated chips were water annealed for 24 h as previously described 38 to induce β-sheet formation and thus film stability in aqueous environments, and subsequently PIII treated as described above. Binding of bovine serum albumin (BSA) to silk surfaces under flow conditions was analyzed by a SPR system (BIAcore 2000, GE Healthcare) at 25 °C as previously described. 39 Briefly, the sensor chip flow channels were washed with PBS at 5 µl/min until a stable baseline was achieved. The flow channels were then exposed to BSA (100 µg/ml in PBS) at a flow rate of 5 µl/min for 8 min and washed with PBS until stable signal was observed. To investigate if the BSA was bound via covalent binding, 3% (wt/v) SDS was flowed over the surface at a flow rate of 5 µl/min for 2 min. The baseline values were subtracted from the binding resonance unit (RU) readings.

2.10. Cell interactions with PIII-treated silk films Bovine Arterial Endothelial Cells (BAECs) were maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Aldrich) supplemented with 3.7g/L sodium bicarbonate, 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) penicillin-streptomycin (Gibco). The media was replaced every 3-4 days and cells were passaged every seven days. For cell culture studies, silk films were cast by pipetting 10 µl of 2% wt/v silk solution onto 5 mm diameter Thermanox coverslips (ProSciTech) and allowing the films to air dry overnight at room temperature. The films were water annealed overnight at room temperature as previously described 40 to induce β-sheet formation and thus film stability in aqueous environments. Samples were PIII-modified as described above for 800 s. Silk films were placed in non-binding 96-well plates (Greiner Bio-One) and 1 × 104 BAECs seeded per well in basal media (no fetal bovine serum) for 1h. Wells were washed three times with phosphate buffered saline (PBS) and cell adhesion, morphology, and proliferation assays performed. Cell adhesion was quantified using the CyQuant assay (Life Technologies) according to manufacturer’s instructions at 1 h post-seeding. Cell adhesion on untreated and PIII-treated silk was compared to that on tissue culture plastic (TCP, positive control) and denatured (80°C, 10 min) 1% (w/v) bovine serum albumin-coated TCP (bTCP, negative control). Following cell adhesion for 1 h, basal media was replaced with DMEM with FBS and cell proliferation quantified over a seven-day period using the Alamar Blue assay (Life Technologies) according to manufacturer’s instructions. Cell proliferation on untreated and PIII-treated silk was compared to that on TCP (positive control) and bTCP (negative control). To evaluate cell morphology, cells were fixed with 4% wt/v paraformaldehyde for 30 min at 37°C, permeabilized with 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 2 mM HEPES, 0.5% v/v Triton X-100, pH 7.2 for 5 min at 4°C and blocked in 1% wt/v BSA in PBS with 0.05% (w/v) Tween-20 (PBST) for 1 h at room temperature. Samples were rinsed with PBST and stained with rhodamine phalloidin (Life Technologies) for 1 h at 37°C and rinsed with PBST. Samples were mounted with

ACS Paragon Plus Environment

7

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 33

Fluoromount aqueous mounting medium and visualized with a Leica TCS SP2 confocal microscope using a 40× objective.

2.11. Statistical analyses Data are expressed as mean ± standard deviation (SD). Statistically significant differences were determined by one- or two-way analysis of variance (ANOVA) and the Tukey post-test. Statistical significance was accepted at p