Composite biodegradable biopolymer coatings of silk fibroin – Poly(3

Applied Surface Science 355 (2015) 1123–1131

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Composite biodegradable biopolymer coatings of silk fibroin – Poly(3-hydroxybutyric-acid-co-3-hydroxyvaleric-acid) for biomedical applications Floralice Marimona Miroiu a , Nicolaie Stefan a,∗ , Anita Ioana Visan a , Cristina Nita a , Catalin Romeo Luculescu a , Oana Rasoga b , Marcela Socol b , Irina Zgura b , Rodica Cristescu a , Doina Craciun a , Gabriel Socol a a b

National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, Magurele, Ilfov, Romania National Institute of Materials Physics, 105 bis Atomistilor Street, Magurele, Ilfov, Romania

a r t i c l e

i n f o

Article history: Received 24 March 2015 Received in revised form 1 July 2015 Accepted 17 July 2015 Available online 22 July 2015 Keywords: Degradability Silk fibroin PHBV MAPLE Drug delivery

a b s t r a c t Composite silk fibroin–poly(3-hydroxybutyric-acid-co-3-hydroxyvaleric-acid) (SF–PHBV) biodegradable coatings were grown by Matrix Assisted Pulsed Laser Evaporation on titanium substrates. Their physicochemical properties and particularly the degradation behavior in simulated body fluid at 37 ◦ C were studied as first step of applicability in local controlled release for tissue regeneration applications. SF and PHBV, natural biopolymers with excellent biocompatibility, but different biodegradability and tensile strength properties, were combined in a composite to improve their properties as coatings for biomedical uses. FTIR analyses showed the stoichiometric transfer from targets to coatings by the presence in the spectra of the main absorption maxima characteristic of both polymers. XRD investigations confirmed the FTIR results showing differences in crystallization behavior with respect to the SF and PHBV content. Contact angle values obtained through wettability measurements indicated the MAPLE deposited coatings were highly hydrophilic; surfaces turning hydrophobic with the increase of the PHBV component. Degradation assays proved that higher PHBV contents resulted in enhanced resistance and a slower degradation rate of composite coatings in SBF. Distinct drug-release schemes could be obtained by adjusting the SF:PHBV ratio to controllably tuning the coatings degradation rate, from rapid-release formulas, where SF predominates, to prolonged sustained ones, for larger PHBV content. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The successful treatment of diseases strongly depends on the efficacy of the therapeutic formulations, while the biomaterials involved in pharmaceutical systems have to be biocompatible, biodegradable, inexpensive and easy to process. Research in the area of drug delivery aims for the development of targeted delivery, so that drugs will be active only within the specified affected areas of the body, with the minimization of adverse effects and, together with sustained release formulations, where the drugs are controllably released in time. Polymer carriers have several advantages over other delivery agents [1]. Silk fibroin (SF), the main constituent of the natural silk, is a natural biopolymer and a fibrous protein produced by silkworms or

∗ Corresponding author. E-mail address: stefan.nicolaie@inflpr.ro (N. Stefan). http://dx.doi.org/10.1016/j.apsusc.2015.07.120 0169-4332/© 2015 Elsevier B.V. All rights reserved.

spiders. SF is the strongest and toughest natural fiber, with unusual high tensile strength and elasticity and proved to be both biocompatible and biodegradable [2–10]. It is subject to prolonged biological proteolytic degradation into easily absorbed aminoacids and is very slowly resorbed in vivo (around one year or more) [3]. Due to the robust and controllable mechanical features, it can be processed under ambient temperature condition or up to 200 ◦ C. It possesses also high transparency, relatively easy functionalization, light weight, and low price; this ancient material becoming a modern attractive option in the fields of biomaterials and tissue engineering, as biocompatible matrix for in vivo applications, functional coatings, as well as candidate for a variety of extremely challenging applications: colorimetric sensors [11], gate dielectric in flexible organic thin-film transistors [12,13], e-paper, dissolvable films for ultrathin conformal bio-integrated electronics, ophtalmological or neurology-compatible applications [14–18]. Polymers as SF, with high amounts of carboxylic acid and weak bonding (hydrogen bonds), instead of strong covalent bonding, are

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suitable for drug delivery. Due to the amino acid sequence, SF offers opportunities for chemical modification with distinct functional groups for easy entrapment of organic molecules or bioactive agents (drugs, growth factors, etc.). Furthermore, bioadhesive polymer carriers, like SF, improve the absorption of drugs in almost any region with epithelial cells, close to the mucosa. On the other hand, structural characteristic of the ␤-sheet configuration similar to viruses was shown to enhance the SF efficiency as drug delivery vehicles [19]. Nonthrombogenic, antiinflammatory, cell adhesive, high contents of short side chain amino acids and exhibiting strong affinity to polysaccharides, aqueous processing and swelling properties that depend on the solution’s pH; all these outstanding properties make SF a unique candidate for controlled and sustained drug delivery [20–28], either as films, nano- or micro-particles. Besides, as recent studies confirmed, SF exhibits an outstanding ability to stabilize immobilized proteins or smaller molecules via distinct or coupled mechanisms: covalently coupling, adsorption, entrapment or encapsulation [29]. Regenerated SF is widely processed into films, gels, scaffolds and microspheres. Fibroin degradation rates depend on its crystalinity, inherently limited [30] and strongly influenced by the processing conditions [3,8]. SF may be combined with other suitable (bio)polymer in order to further extend the range of adjustable drug release systems. Polyhydroxyalkanoates or PHAs are natural biodegradable and bioresorbable linear polyesters bacterially synthesized and accumulated as an intracellular carbon and energy storage compound. PHA polymers are UV stable, thermoplastic, can be processed on conventional processing equipment, show low permeation of water (good resistance to moisture) and aroma barrier properties [31–33]. Depending on composition, they are ductile and more or less elastic, with the crystallinity varying up to 70%. Poly-3-hydroxybutyrate (PHB), as well as its copolymers with 3-hydroxyvalerate (3HV), poly(3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV), are the most known representatives of PHA family. They are considered biomaterials suitable for tissue engineering, medical sutures, cardiovascular stents and drug delivery [32], due to their biocompatibility, biodegradability, nontoxicity and thermoplasticity properties [31–33]. Long-term stable in air and moisture, PHBV is however biodegraded by the active microbial environments into HB and HV fragments, which are viewed by the cells as carbon sources for growth [34]. Maybe this interesting aspect could prove beneficial in particular cases of targeted drug delivery. Most notably, the degradation products of PHB and PHBV are natural metabolites present in blood [35,36]. Moreover, considering that PHBV degrades rather slowly, it may be thought as a long-term delivery carrier and/or favoring the bone growth and healing in different medical applications, where it would finally avoid replacement surgery [31,33,36]. Different types of PHBV, in terms of chemical composition (copolymers’ ratio) and synthesis method, vary in surface properties, such as surface roughness and hydrophilicity, important in clinical applications as those mentioned by other authors [36–38]. Mechanical properties, like toughness, impact strength, flexibility and processability, improve with a higher percentage of hydroxyvalerate in the material [31,33,34]. In the present study we aimed to synthesize mixed SF–PHBV coatings, taking advantage of all the outstanding properties of the two biopolymers and obtaining tuned degradability, for attending targeted biomedical uses. Matrix Assisted Pulsed Laser Evaporation (MAPLE) is a non-contact, advanced laser technique designed to produce safe and accurate transfer in form of thin films of organic and polymeric materials, without fragmentation and preserving their functions [39–55]. It is also able to transfer dopants, offers a good adherence, a precise thickness control, layering and patterning ability, the compatibility with different substrates materials

and with the computer aided manufacturing [39–45]. MAPLE offers many advantages with respect to the techniques that require dissolution of polymers in solvents and intimate wetting of polymer solutions applied to the substrate to be coated, as for the latter ones the solvent controls the mobility of the polymer on the substrate surface and may generate non-uniformities [42]. Our previous studies indicated a positive behavior of bone cells cultured on the hydroxyapatite-fibroin as thin films deposited by MAPLE [56]. Though it may be also used to synthesize polymeric nano- or micro-spheres [44,57,58], MAPLE could be an excellent choice to deposit polymer (in our case SF or SF-based) continuous coatings delivering therapeutic agents [59–61], useful in case of small coated orthopedic implants, or stents, where both biocompatibility and additional sustained drug release (usually involving biodegradability) are needed. It allows a rather rapid preparation of SF or SF-based solutions or suspensions and does not require any minimal surface charge of the substrate, which could deform the molecules, as other layer-by-layer methods. This study reports on the synthesis and degradation behavior of composite silk fibroin–poly(3-hydroxybutyric-acid-co-3hydroxyvaleric-acid) (SF–PHBV) polymeric coatings as mandatory step prefacing any study on the effective drug delivery ability or biological functionalization of such coatings. In this view, all the deposited SF–PHBV composite coatings, as well as their simple constituents were tested in terms of wettability and degradation in dynamic flux of simulated body fluid (SBF).

2. Materials and methods In our experiments we used commercial 2 ␮m granulation powder of degummed (i.e. sericin-free) Bombyx mori silk fibroin (Wuxi Smiss Technology Co., Ltd., China), and commercial Sigma Aldrich flakes of poly(3-hydroxybutyric-acid-co-3-hydroxyvalericacid) with PHV 8% molar, natural origin, respectively. Commercial pure (CP) grade 4 Ti disks of 12 mm in diameter were provided by Tehnomed SA. Suspensions of 3:1, 1:1, 1:3 SF–PHBV weight ratios, as well as simple SF or PHBV in chloroform (Merck, grade purity 99%) with 30 g/L (w/v) concentration were prepared to synthesize the MAPLE targets. Before freezing, their homogeneity was kept by magnetic stirring. The solid MAPLE targets were obtained from these solutions frozen in liquid nitrogen and kept under the melting point during deposition process by means of a cooler continuously filled with liquid nitrogen. An excimer laser source KrF* ( = 248 nm, pulse duration = 25 ns and 15 Hz pulses repetition rate) was used for all the reported MAPLE depositions. Three values of laser fluence were chosen at 0.3, 0.4 and 0.5 J/cm2 , while the pressure inside the vacuum chamber was kept at 1 Pa. A laser beam homogenizer was used to improve both the energy distribution in the laser spot and the deposition area on the substrate. During deposition, the target and the substrate were rotated to avoid the target piercing and to ensure a better uniformity of the grown films. Series of subsequent 10 000 (for physico-chemical investigations) or 40 000 (for degradation tests) laser pulses were applied for the thin or thicker deposited coatings, depending on their destination, as further mentioned. The thickness of these coatings assessed by a Stylus Profiler XP2 system (Ambios Technology, Santa Cruz, CA, USA) was between 2 and 5 ␮m, function of their experimental purpose, physical or degradation analyses. The deposition substrates, 10 mm2 glass coverslips or doubleside polished Si (1 0 0) transparent in the IR, were placed at 5 cm distance parallel to the target and maintained at RT during the MAPLE synthesis process. Polymeric coatings aimed for degradation studies were deposited on grade 4 titanium disks of 12 mm

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Fig. 1. Injection multichannel degradation reactor setup.

Fig. 3. Typical XRD diffractograms for SF–PHBV composite, SF and PHBV coatings.

Fig. 2. FTIR spectra of (a) 1:1 SF–PHBV dropcast and films deposited at 0.3, 0.4 and 0.5 J/cm2 fluences; (b) SF, PHBV and 3:1, 1:1, 1:3 SF–PHBV composite coatings deposited by MAPLE at the selected 0.3 J/cm2 laser fluence.

diameter. Prior to deposition, all the substrates were carefully cleaned in an ultrasonic bath in acetone, ethanol and deionized water and blow-dried with N2 gas. For comparison data, dropcast samples were prepared by uniformly spreading drops of polymeric

solutions on the IR transparent (1 0 0) both sides polished silicon or glass slides. The surface morphology of the deposited films was investigated by scanning electron microscopy (SEM) by means of an Inspect S Scanning Electron Microscope (FEI, Inc., USA) with 20 kV accelerating voltage, and 250–20 000× magnification. The samples were capped with a thin Au film in order to reduce electrical charging. Fourier transform infrared (FTIR) spectroscopy study was conducted with a Shimadzu 8400S Spectrometer, in transmission mode. The spectra were recorded in the 4000–550 cm–1 range, with a resolution of 4 cm–1 and series of 50 scans per sample. The crystalline status of the deposited coatings was assessed by X-Ray Diffraction (XRD) using a Bruker D8 Advance diffractometer equipped with a Cu target X-ray tube, in parallel beam setting. The scattered intensity was recorded in the range 20–50◦ (2), with a step size of 0.04◦ and 6 s per step. The wettability properties of the analyzed samples were determined by measuring at room temperature the static contact angle (CA) with a Drop Shape Analysis System (model DSA100, Kruss GmbH, Germany) and averaging the values of three measurements for each coating. Samples were placed on a plane stage, under the tip of a water-dispensing disposable blunt-end stainless steel needle with an outer diameter of 0.5 mm. The fixed needle was attached to a syringe pump, which was controlled by the computer for drop delivery [62]. The volume of the drops was of ∼2 ␮L. The CAs were

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Table 1 Contact angle and the mass loss of polymeric coatings deposited at 300 mJ/cm2 laser fluence. Samples type

Composition

Wettability contact angle (◦ )

SF 3:1 SF–PHBV 1:1 SF–PHBV 1:3 SF–PHBV PHBV

Silk fibroin 3:1 Composite 1:1 Composite 1:3 Composite PHBV