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Gradient multifunctional biopolymer thin film assemblies synthesized by combinatorial MAPLE
Applied Surface Science 466 (2019) 628–636
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Full Length Article
Gradient multifunctional biopolymer thin film assemblies synthesized by combinatorial MAPLE
T
Natalia Mihailescua, Merve Erginer Haskoylub, Carmen Ristoscua, Müge Sennaroglu Bostanc, Mihai Sopronyia,d, Mehmet S. Eroğluc,e, Mariana Carmen Chifiriucf, ⁎ Cosmin Catalin Mustaciosug,h, Emanuel Axentea, Ebru Toksoy Onerb, Ion N. Mihailescua, a
National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, Magurele, Ilfov RO-77125, Romania Department of Bioengineering, Marmara University, Goztepe, 34722 Istanbul, Turkey c Department of Chemical Engineering, Marmara University, Goztepe, 34722 Istanbul, Turkey d Physics Department, University of Bucharest, Magurele, Ilfov RO-077125, Romania e TUBITAK-UME, Chemistry Group Laboratories, 41471 Gebze, Kocaeli, Turkey f Department of Microbiology, Faculty of Biology and Research Institute of the University of Bucharest (ICUB), University of Bucharest, 060101 Bucharest, Romania g Horia Hulubei National Institute for Physics and Nuclear Engineering IFIN-HH, Magurele, Ilfov RO-077125, Romania h Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Romania b
A R T I C LE I N FO
A B S T R A C T
Keywords: Combinatorial-MAPLE Biopolymer coatings Compositional maps Sulfated Halomonas Levan - quaternized low molecular weight Chitosan mixtures Anti-biofilm activity
Combinatorial Matrix-Assisted Pulsed Laser Evaporation (C-MAPLE) was recently introduced to the fast generation of compositional libraries of two biopolymers in a single-step process, for tissue engineering and regenerative medicine applications. Synchronized laser irradiation of two distinct cryogenic targets, one consisting of Sulfated Halomonas Levan and the other of quaternized low molecular weight Chitosan was used to fabricate compositional gradient coatings for surface functionalization. Synthesized coatings preserved the base material composition as confirmed by Fourier Transform Infrared Spectroscopy. Morphological study by Scanning Electron Microscopy, Atomic Force Microscopy and profilometry correlated with water contact angles measurements demonstrated that the obtained thin coatings have improved surface properties with respect to pure material coatings. Fluorescence microscopy validated the compositional gradient, while in vitro assays evidenced characteristic responses of mouse fibroblasts (L929 cell line) by distinct deposition surface regions. The coagulation test pointed out good properties for Sulfated Halomonas Levan coatings as compared to the case of an increased amount of quaternized low molecular weight Chitosan biopolymer or the control. The antimicrobial effect of the coatings was demonstrated against Escherichia coli and Staphylococcus aureus strains, representative for both Gram negative and Gram positive bacterial species, respectively, mainly involved in implant and nosocomial infections. The assembled nanostructures possess variable anti-biofilm activity along the compositional gradient, with a stronger inhibitory effect on the initial adherence phase of both tested microbial strains, but also against mature Escherichia coli biofilms. It was shown that C-MAPLE can generate discrete areas of blended polymeric composition exhibiting improved surface properties for a broad range of biomedicine applications, e.g. the fabrication of thin bioactive and cell-instructive coatings with anti-adherence properties.
1. Introduction Recently, functional coatings attracted a rapidly growing interest, due to the possibility to explore novel properties of materials at either micro- or nanoscale. With respect to their original protective function, thin films are nowadays in the forefront of sustained advances in
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fabricating antimicrobial, bioactive and biomimetic, transparent and conductive, thermo-electric, self-healing, self-cleaning and super-hydrophobic coatings, to mention a few applications only. On the other hand, combinatorial research, in its whole spectrum of applications in materials science and chemistry, has proven to drive fast progress in the synthesis of innovative materials or new properties for nanomedicine.
Corresponding author. E-mail address: ion.mihailescu@inflpr.ro (I.N. Mihailescu).
https://doi.org/10.1016/j.apsusc.2018.10.077 Received 7 June 2018; Received in revised form 5 October 2018; Accepted 8 October 2018 Available online 09 October 2018 0169-4332/ © 2018 Elsevier B.V. All rights reserved.
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In the biomedical field, most of the applications are related to the fabrication of bioresponsive surfaces and interfaces able to modulate cells behavior and push forward drug discovery and delivery systems [1,2]. Indeed, combinatorial techniques, correlated with highthroughput screening stand for the best tools to improve the composition-structure and/or properties relationship for both organic and inorganic library coatings [3]. We have recently introduced a single-step combinatorial approach based on Matrix-Assisted Pulsed Laser Evaporation (MAPLE) for both the immobilization and blending of organic and environmental friendly polymeric compounds [4–8]. Later, both experimental and theoretical aspects of C-MAPLE technique were extensively addressed in our studies of different organic, inorganic or composite materials [9,10]. Laser-based synthesis of biopolymers combinatorial coatings is reviewed in Ref. [9]. Chemically modified or natural polysaccharides with sulfate groups have efficient biological characteristics, such as antioxidant and immunomodulation [11,12], antiherpetic [13], antithrombotic and anticoagulant [14,15] action. Levan is a β-2, 6-linked fructose homopolymer synthesized extracellularly from sucrose substrates by various microorganisms. Due to its unique features, it is considered a quite valuable biomaterial with many potential applications in medical, food, pharmaceutical, cosmetics and chemical industries [16]. The Gram negative aerobic bacterial strain Halomonas smyrnensis AAD6T was reported as the first halophilic levan producer [17], besides its antioxidant and anticancer activities [18,19]. Halomonas Levan (HL) was proved suitable for use as drug carrier systems [20], bioactive thin film blends [21], multilayer adhesive films [22], but also temperature responsive [23] and cytocompatible hydrogels [24]. Recently, a sulfated derivative of HL (SHL) was reported as a heparin mimetic anticoagulant [4,14]. Also, electrospun matrices of SHL with anticoagulant activity were found to have high potential to be used in decreasing neointimal proliferation and thrombogenicity of grafts and prosthesis [25]. SHL was shown to improve the mechanical and adhesive properties of multilayered free-standing films and allow myogenic differentiation and lead to cytocompatible and myoconductive films for cardiac tissue engineering applications [26]. Recent studies reported the characteristics of HL and oxidized HL (OHL) thin films synthesized by MAPLE [27]. HL and OHL were combined and binary gradient films were fabricated using the Combinatorial MAPLE (C-MAPLE) technique. In vitro cell culture studies with the SaOS2 bone cell line have shown that the osteoblasts’ extracellular signal-regulated kinase signaling was modulated with different propensity. It was suggested that C-MAPLE could indeed serve as a suitable method for the fabrication of new bioactive surfaces controlling the cell response [5]. Chitosan (CH) is the deacetylated product of the well-known natural biopolymer chitin. Its chemical reactivity is attractive due to the presence of –NH2 groups [28]. Chitin and Chitosan are highly basic unlike most other natural polysaccharides [29]. Chemical properties of CH include the linear polyamine structure, reactive amino and hydroxyl groups and possibility to chelate transitional metal ions. CH is a natural, biocompatible, biodegradable, safe, non-toxic polymer [30,31]. It has hemostatic, anti-thrombogenic, antibacterial, fungistatic, spermicidal, immunoadjuvant, antitumor, anti-cholesteremic biological properties and may accelerate bone formation and so can be used as central nervous system depressant [32,33]. Because of diverse bioactivities, CH can be applied in a wide range of biomedical applications such as wound healing or tissue engineering [31,34], implant coatings [35–38] and drug delivery systems [28,31,39,40]. CH molecular weight exerts a major influence upon its biological and physical-chemical properties. Thus, the crystallinity, degradation, tensile strengths and moisture content have been shown to be dependent on the molecular weight [30,41–44]. As known, commercially available CH has a high molecular weight. Accordingly, methods have been developed using ultrasound, heat, enzymatic or chemical
hydrolysis to depolymerize CH. Depolymerization of CH using nitrous acid (HNO2) was imposing as a preferred technique, because it is rapid, cost-effective and can be monitored to produce CH of a pre-selected size [45–49]. The way to improve or add new properties to CH [50] is to chemically modify the chain by adding functional groups to the main one, non-altering the original structure and properties. The primary amine and hydroxyl groups (OH) are responsible for reactions such as e.g. quaternization. Moreover, the presence of these reactive primary amino groups adds special properties to CH which make them very appropriate in pharmaceutical applications [51–53]. There are several studies in literature on CH quaternization, reporting the improvement, even under basic conditions, of properties such as the antimicrobial activity [53–56], moisture-retention capacity [57,58], bio-adhesivity, permeation enhancing effect and antifungal activity [56,58]. The possibility to deposit two natural, biocompatible, non-toxic polysaccharides, i.e. Sulfated Halomonas Levan (SHL) and quaternized highly deacetylated low molecular weight CH (QCH) on Si, glass or Ti substrates is explored in this study. We mainly aimed to fabricate thin coatings able to offer a suitable interface for cells leading to an improved osseointegration and simultaneously exhibiting good antimicrobial properties. To this purpose, C-MAPLE method was applied to transfer and deposit SHL and QCH in a single process in order to produce new organic thin libraries with improved properties. To the best of our knowledge, this is a first attempt to synthesize in situ SHL and QCH thin coatings for the selection of best bioactive surface able to control cells behavior and ensure protection against microbial colonization and biofilms formation. 2. Materials and methods 2.1. Synthesis of combinatorial functional coatings by C-MAPLE The preparation of quaternized highly deacetylated and low molecular weight CH (QCH) was carried out via established protocols described in literature [21,40,49,58–60] (and given in detail in Supplementary Information (SI) section). The sulfonation of Halomonas Levan (SHL) was achieved following the procedure described in Refs. [14,25,26] (also detailed in SI). First, 25 mg of SHL were dissolved in 5 mL of dimethyl sulfoxide (DMSO) to obtain a homogeneous solution. DMSO was selected as solvent because it does not chemically interact with SHL, and efficiently absorbs the laser wavelength (248 nm) used for the evaporation of the target in frozen state [27]. A concentration of 2% QCH dissolved in deionized H2O (dH2O), was used for the preparation of the second CMAPLE target. The two solutions were transferred into distinct compartments of a ring-like concentric copper holder. This holder has been designed and manufactured to accommodate in each compartment between 5 and 10 mL of solution to be used in C–MAPLE experiments. The holder containing the solutions was carefully immersed in liquid nitrogen (LN) to get the two cryogenic targets. Next, the holder was mounted inside the cooler, which was under the continuous supply of LN, to keep the target frozen. The schematic representation of the C-MAPLE experimental set-up is depicted in Fig. 1. The laser beam generated by a KrF* excimer laser source (λ = 248 nm, τFWHM = 25 ns), operating at a repetition rate of 10 Hz is divided by an optical splitter. The two beams are directed and focused onto the two concentric targets. The distance between the centers of the two laser spots is set at 2 cm. The evaporated polymers were collected either onto glass, Si (1 0 0) or Ti substrates placed parallel to the targets, at a separation distance of 5.5 cm. Before deposition, all substrates were successively cleaned in an ultrasonic bath for 15 min in acetone, alcohol and dH2O. For the growth of each SHL to QCH coatings, 40.000 laser pulses have been applied. One obtains in this configuration the polymer co-deposition onto a 4 cm long sample, the edges containing 100% SHL at one end and 100% QCH at the other one. A continuous gradient of SHL–QCH blended composition is reached in 629
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by using an UV lamp in a biological cabinet for one hour. 2.3.1. Viability/MTS assay CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay – MTS (Promega) was used for the viability evaluation of L929 mouse fibroblasts (cell line between passages 11 and 14). The cells were detached with trypsin/EDTA (Biochrom) and resuspended in complete medium – MEM with 10% fetal bovine serum (FBS) and L-glutamine at a density of 200,000 cells per mL. Cells were cultured for 30 min in an incubator, so that to firmly adhere to coatings surface. After 24 h incubation, the coatings were moved in new wells. 400 µL of MTS solution were added in MEM with 5% serum and cells were incubated again for 3 h. 100 µL of medium from each well was poured in a 96-well plate, for an absorbance measurement at 490 nm. 2.3.2. Cell morphology examination Cells morphology was assessed using fluorescence microscopy imaging (Olympus BX51fluorescence microscope). The cells were fixed after 24 h incubation using 4% paraformaldehyde, permeated with 0.1% Triton X and incubated overnight at 4 °C with Fluorescein Phalloidin (Thermo Fisher). Hoechst stain (Thermo Fisher) was added for nuclei visualization and the samples were mounted on the slide with ProLongGlodantifade reagent (Thermo Fisher).
Fig. 1. C-MAPLE experimental setup.
between [5,7,8].
2.3.3. Coagulation test Coagulation test (clotting assay on microscope slide) was used for evaluation of the anticoagulant properties of deposited coatings as compared with controls (glass slide). Venous blood from three clinically healthy men, aged 25, 30 and 33 was collected and mixed in equal proportions with Phosphate Buffered Saline (PBS), homogenized and deposited into droplets on coated surfaces. The coatings were previously placed in a Petri dish on a bed of wet tissue to avoid drying. The plates were inclined slightly from time to time to observe the coagulation of the blood with the formation of clot. When the drop stopped moving it was considered that the blood coagulated. Coagulation experiments were performed at RT.
2.2. Physical–chemical characterization of thin gradient coatings The chemical composition of the thin films libraries was studied by Fourier Transform Infrared (FTIR) Spectroscopy with a Shimadzu 8400S FTIR apparatus. Spectra were recorded within the range 4000–500 cm−1, with a resolution of 8.0 cm−1 and a total of 80 scans/ experiment. The measurements were performed along the longitudinal direction of the thin gradient coatings (x direction in Fig. 1). Scanning Electron Microscopy (SEM) was used to examine the surface morphology of the deposited coatings with a FEI Inspect S electron microscope. The measurements were carried out at 20 kV acceleration voltages, in high vacuum, under secondary electrons acquisition mode. Before SEM, the samples were coated with a very thin layer of gold. Atomic Force Microscopy (AFM) with a Workshop TT-AFM apparatus was performed for surface roughness investigations. Images were acquired from different areas of each coating in non-contact mode. Profilometry analysis was done for thickness investigations with an Ambios XP-2 apparatus for a measurement length of 1.5 mm, weight 5 mg, speed 0.01 mm/sec. A step between substrate and thin films was imprinted on each sample and the measurements were carried out on three distinct points for each coating. For statistics, measurements were carried out on three identical samples. SHL and QCH optical and fluorescence microscopy images were acquired with a DM 4000B LED, Leica microscope. Twin images were captured along the C-MAPLE generated SHL–QCH gradient keeping the same exposure time for detection of fluorescence emission between specimens. Contact Angle (CA) measurements by sessile drop technique in static mode were conducted to inspect the hydrophobic/hydrophilic features of the coatings. A Krüss DSA-100 model CA meter equipped with High Performance Frame Grabber Camera T1C (25 frames per second) was used with DS3210 software controlled auto-dosing system. Drops of 5 µL of dH2O were deposited on a dry and clean surface using the autodosing system. Static measurements were conducted immediately after the sessile drop formation and at least five measurements were performed on each sample, at ambient temperature and humidity. The results were statistically averaged.
2.3.4. Anti-biofilm activity assay The anti-biofilm activity of the obtained coatings was carried out with Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8739 strains. The tested coatings were previously sterilized by exposure to UV for 30 min. After UV sterilization, an amount of 20 µL of microbial suspension of 0.5 MacFarland density prepared from fresh cultures developed on tryptic soy agar (TSA) were distributed over the coatings treated surface and incubated at 37 °C in humid atmosphere for 8 or 24 h. Immediately after deposition and after each incubation period, the coatings were suspended in 5 mL sterile saline, vortexed vigorously to resuspend the adherent bacteria. Serial ten-fold dilutions were performed from the recovered suspension, spotted on solid medium in triplicate spots of 10 µL each and the viable cells counts were determined and expressed as colony forming units (CFU)/mL. 3. Results and discussion 3.1. Pure biopolymers FTIR spectrum of QCH (Fig. 2) revealed the presence of all characteristic bands. This is supported by the broad eOH and sharp NeH absorption peaks of CH at 3400 and 3100 cm−1, and CeH bond in eCH2 (ʋ1 = 2920 cm−1) and eCH3 (ʋ2 = 2875 cm−1) groups, respectively [58]. Peaks observed in the range of 1200–1000 cm−1 are assigned to the CeOeC bending vibration of glucose rings and glucose amine bonds, proving that the main chain of CH was not decomposed by oxidation and quaternization. The peak at 1548 cm−1 is assigned to the NeH bending vibration of glucose amine groups. The peaks at 1641 and 681 cm−1 belong to the NeC bending vibration of
2.3. Biological tests Prior to in vitro assay, all coatings and glass controls were sterilized 630
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Fig. 3. FTIR Spectra along the thin film from Levan (SHL) to Chitosan (QCH).
Fig. 2. FTIR spectrum of QCH powder.
3.2.2. SEM and profilometry The surface morphology of SHL, SHL/QCH, and QCH/SHL and QCH coatings is visible in Fig. 4. The coatings consist of a homogenous structure along longitudinal axis of the glass slide from SHL to QCH regions, as revealed by higher magnification in Fig. 4 bottom row. One may notice the display of feature characteristics to each compound. However, at macroscale (Fig. 4 top row), an irregular but interconnected polymer network of quasi-spherical particles is evidenced in the case of SHL, which decrease with QCH content in the combinatorial samples. The profilometry measurements converged to thickness mean values of: ∼450 nm with a mean Standard Deviation (SD) of ± 0.37 SD for SHL, ∼320 nm ± 0.17 SD for SHL/QCH, ∼350 nm ± 0.21 SD for QCH/SHL, and ∼200 nm ± 0.13 SD for QCH coatings. One can estimate on this basis the following growth rate for each coating of: SHL = 0.007; SHL/QCH = 0.011; QCH/SHL = 0.009 and QCH = 0.005 Å/laser pulse.
glycidyltrimethyl ammonium chloride (GTMAC), and stand for a clear evidence of CH quaternization [59]. Additionally, the band visible at around 1475 cm−1 is attributed to the eCH3 groups of GTMAC as confirmed by the study in Ref. [60]. Deacetylation degree was taken as 98% by 1H NMR spectroscopy, in agreement with Refs. [21,40,61], while the conversion of quaternization reaction was found as 28% of the glucosamine rings (see Fig. S2 in SI for details). Molar mass values of CH, DCH and QCH were estimated by online Gel permeation chromatography (as described in SI) and collected in Table 1.
3.2. Characterization of deposited coatings 3.2.1. FTIR Composition was inferred from infrared absorption spectra (Fig. 3) recorded for four representative zones from SHL to QCH thin C-MAPLE coatings. One mentions that the absorption band of HL at 3350 cm−1 is characteristic to the eOH stretching of fructofuranose rings and eCH2 OH groups, respectively. CeH stretching vibration of fructose residues group is supported by the appearance of the two peaks at around 2920 and 2873 cm−1. The weak absorption peak visible at ∼930 cm−1, together with the sharp bands at 1040 and 1130 cm−1 are assigned to the CeOeC symmetric bending vibration of fructofuranose rings and glycosidic linkages [62]. The absorption bands peaking around 1203 and 830 cm−1 are attributed to SHL and support the asymmetric stretching vibration of S]O linkages and symmetrical CeOeS vibration of CeOeSO3 groups, respectively [14,63]. The intensity decrease of all specific absorption bands of SHL is visible and assigned to QCH blending. The major absorption bands of QCH, occurring at 1646, 1588 and 1324 cm−1, are characteristic to eC]O vibration of Amide I, NeH bending, eNH2 to Amide II, and NeH bending to Amide III, respectively. The CeOeC bending vibration of glucose rings and glycosidic linkages appear at 1076, 1021 and 972 cm−1. FTIR investigation convincingly demonstrated the gradient composition from SHL to QCH obtained by C–MAPLE.
3.2.3. AFM High resolution topographic images for SHL to QCH coatings deposited by C-MAPLE were acquired by AFM (Fig. 5). Image analyses revealed that SHL coating exhibits the following roughness parameters: Ra = 11.2 nm and Rms = 14 nm. Nanoparticles evenly spread out over the entire scanned area have dimensions ranging from below 100 nm to 800 nm. The SHL/QCH image is characterized by roughness parameters of Ra = 23.8 nm and Rms = 38.5 nm. An increased magnification indicates the presence of QCH nanoparticles with dimensions of ∼100 nm, which are well visible. They are closely packed with a tendency to form a continuous coating. The largest particles with diameter of about 600 nm are placed near the center of the coating. They are the most probably clusters of material forming during deposition. In case of QCH/SHL coating the roughness parameters increase to Ra = 101 nm and Rms = 140 nm exhibiting higher magnification details (∼400 nm) as compared to SHL and SHL/QCH regions. Nanoparticles with a dimension within the 50 ÷ 500 nm range are more compactly packed in case of QCH and QCH/SHL vs. SHL and SHL/QCH coatings, suggesting a clear clusterization tendency (Fig. 5). Islands of nanoparticles start forming, as visible in the center of the image. AFM investigations of simple QCH coating evidence roughness parameters: Ra = 91.2 nm and Rms = 118 nm, exhibiting lower magnification details (∼200 nm) than QCH/SHL coating. Nanoparticles of 100 ÷ 300 nm diameter are visible agglomerated in clusters which are assembling in form of irregular islands with dimension of 2 ÷ 5 µm (the lighter areas) separated by lower magnification areas (the dark ones). The average magnification of the topography is ∼300 nm. By correlating AFM and SEM investigations, one may conclude that
Table 1 Number Average molecular weight (Mn), Weight Average molecular weight (Mw) and polydispersity index (PDI) values for original (CH), deacetylated (DCH) and quaternized (QCH) chitosan. Sample
Mn (g/mol)
Mw (g/mol)
Mw/Mn (PDI)
CH DCH QCH
9.475e+4 (0.4%) 7.319e+4 (0.4%) 5.383e+3 (3%)
1.670e+5 (0.3%) 1.177e+5 (0.3%) 7.023e+3 (3%)
1.763 (0.5%) 1.608 (0.5%) 1.305 (5%)
631
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Fig. 4. SEM micrographs of SHL (left) to QCH (right) gradient coatings prepared by C-MAPLE. The images were collected at two magnifications: top row is at 1000x magnification with a scale bar of 50 µm and bottom row is at 20,000x magnification with a scale bar of 4 µm.
QCH samples exhibit more homogenous surface features in respect with SHL coatings. On the other hand, roughness increases with the content of QCH, which has Rms of 118 nm while this is of only 14 nm for SHL surface. Since SHL has a higher molecular weight (> 2 * 106 g/mol; [25]) than QCH, the observed surface roughness difference between samples could be due to distinct chain lengths and polarities between sulfate and tri-methyl ammonium groups. The higher polarity of sulfate groups of SHL provided better contact with the glass substrate which leads to lower roughness. It should be stressed out that the presence of particulates of various shapes all over the gradient coatings is an intrinsic characteristic of this deposition method. One therefore expects benefices due to the increased surface roughness resulting in a better insitu anchorage of the implant. Micro-movements of the medical device are minimal therefore ensuring the required primary immovability till bone grows and attaches to the implant surface. Moreover, these particulates are beneficial for the growth and proliferation of cells because they favour the better anchorage of cells roots or cytoplasmatic extensions [64]. Recently, it was demonstrated that cells respond to nanoscale physical properties of the biomaterial [65]. The nanoscale topography provides superior support for the proliferation of bone cells due to the advantageous interaction between nanosize irregularities on the material surface and adsorbed cell adhesion-mediating molecules [66]. Accordingly, a surface roughness in tens of nanometers range is desired for the adhesion, growth, differentiation and phenotypic maturation of bone cells, rather than flat surfaces or surfaces with submicron- or micro-scale roughness.
Table 2 CA results. Sample
Contact Angle [˚]
Control -glass slide SHL SHL/QCH QCH/SHL QCH
66.1 13.0 85.2 84.1 66.3
than weak carboxyl groups and absorb more water [67]. QCH region points to almost the same CA value as the control sample (Table 2). When SHL is combined with QCH, as e.g. in the central region of the combinatorial coating, one observes that hydrophobicity of both polymers is increasing. Biopolymers blending and assembling on substrate generate more hydrophobic surface features as compared with native ones. According to AFM data, increased roughness is resulting in a lower wettability of the surface. Concerning SHL/QCH and QCH/SHL structures, as neutralization is reached with the opposite charges along with the increase of the roughness, a higher CA was recorded as compared to native polymers. Previous studies demonstrated that MAPLE layers of Oxidized Halomonas Levan behave more hydrophilic than Halomonas Levan as a result of the acidic aldehyde-hydrogen bonds [27]. SHL showed noticeably higher hydrophilicity (CA = 13°) compared to native Levan (CA = 58°) as reported in Ref. [27], due to the presence of sulfonate groups. Moreover, moderate hydrophylicity/hydrophobicity favours cell-substrate interaction [68].
3.2.4. Contact angle (CA) measurements CA results obtained with dH2O sessile drops on control (uncovered glass slide), SHL, SHL/QCH, QCH/SHL and QCH C–MAPLE deposited coatings are collected in Table 2. Images revealed that SHL coating has a highly hydrophilic surface characterized by a CA of 13.0°, in agreement with previous studies [14]. As known, strong sulfonate groups have shorter water residence times
3.2.5. Fluorescence microscopy The fluorescence signal evolution along the coatings from SHL to QCH was investigated to qualitatively evidence the gradient between the two polysaccharides. Paired optical and fluorescence microscopy images of SHL to QCH vs. glass control, are given in Fig. 6a. As visible, an almost linear evolution of QCH fluorescence intensity signal is
Fig. 5. AFM images of investigated areas of SHL to QCH coatings. The SHL and SHL/QCH images were acquired on 5 × 5 µm2 area, and the QCH/SHL and QCH images were collected from a 15 × 15 µm2 area. 256 × 256 lines and 0.5 Hz scan frequency were used. 632
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Fig. 6. (a) Fluorescence microscopy and optical images of SHL–QCH (up and down, respectively) gradient coatings obtained by C-MAPLE along the glass slide. Glass controls are given. Magnification Bar = 100 µm (20×). (b) Schematic representation of composition gradient.
reached (Fig. 6b), which serves as a clear demonstration of the composition gradient, in agreement with FTIR spectroscopy investigations.
Table 3 L929 cells viability on C-MAPLE samples and control, as obtained in 4 different experiments.
3.3. Biological tests Experiment Experiment Experiment Experiment
3.3.1. Viability/MTS assay L929 cells viability on the obtained combinatorial coatings containing SHL-QCH mixtures is slightly larger than the glass control (Fig. 7), promoting a stimulating effect of C–MAPLE library on both the cell attachment and growth.
1 2 3 4
Control
SHL
SHL/QCH
QCH/SHL
QCH
1.171 1.223 1.229 1.211
1.224 1.218 1.196 1.236
1.26 1.269 1.218 1.303
1.374 1.343 1.352 1.392
1.323 1.353 1.376 1.359
From statistical point of view, after at least 3 experiments, particularly in the case of QCH/SHL mixture, the cells viability is reproducible and it is superior to the control (Table 3). Moreover, the combination of biopolymers has significantly increased cells viability, specifically in case of QCH/SHL mixture. It reached 110% of the most used control in literature, clearly demonstrating the biocompatibility of the gradient coatings obtained by combinatorial MAPLE. 3.3.2. Cell morphology Fluorescence microscopy images of L929 cells seeded on C–MAPLE samples and on control show that the cells express normal morphology, with a polyhedral and elongated shape, exhibiting a well-established cytoskeleton on both control and coatings. This is in agreement with the results of the MTS assays. Moreover, the nuclear morphology (areas marked with white circles in Fig. 8) indicates that the cells are in various stages of division with a normal proliferative behavior. This stands for a good indicator of the biocompatibility of the C–MAPLE coatings.
Fig. 7. L929 cells proliferation on SHL, SHL/QCH, QCH/SHL, QCH C–MAPLE coatings and on glass control.
3.3.3. Coagulation Data in Table 4 show that the QCH coatings alone have no 633
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Fig. 8. Fluorescence microscopy and Bright Field images of L929 cells onto control (glass) and on SHL, SHL/QCH, QCH/SHL, and QCH C–MAPLE coatings (magnification × 40). Actin fibers are marked with green by Fluorescein-Phalloidin, while Nucleus is stained with blue Hoechst. Table 4 Anticoagulant activity of SHL, SHL/QCH, QCH/SHL, and QCH coatings. Sample
Control (glass)
SHL
SHL/QCH
QCH/SHL
QCH
Time (min)
8
106
40
15
8.5
significant effect on the clotting time. They are similar to the glass control. On the other hand, the clotting time increases when increasing SHL concentration in C–MAPLE samples. SHL coatings exhibit the highest clotting activity. 3.3.4. Antibiofilm assay We have investigated the antibiofilm effect of the obtained bioactive polymeric coatings against S. aureus and E. coli strains. They were selected as relevant Gram positive and Gram negative microorganisms, respectively, which are the most frequently involved in the etiology of medical devices associated infections. Anti-adhesion and antibiofilm activity of the obtained coatings were tested in triplicate, immediately after the deposition of the microbial suspension on the surface of the samples, and after 8 h or 24 h of incubation. The tested samples exhibited variable antibiofilm activity, depending on the incubation time, the gradient of the two polymers and the microbial strain. The coatings containing the highest amount of levan (SHL) or chitosan (QCH) proved the most effective anti-adhesion activity, quantified immediately after the contact with S. aureus cells (T0). In exchange, once the S. aureus biofilm starts to grow (T1), the efficiency of the obtained coatings decreases, becoming comparable or even lower than that of the uncoated control sample (Fig. 9). In case of E. coli, the initial adherence of the cells quantified at T0 was inferior in case of all types of polymeric coatings, as compared to
Fig. 10. Temporal dynamics of the E. coli biofilm growth on the obtained coatings.
control. After 8 h of incubation (T1) all tested coatings proved a lower antibiofilm activity than the uncoated control sample. In exchange, at 24 h of incubation (T2), all coatings, and particularly the samples SHL/ QCH and QCH/SHL proved a very intensive antibiofilm activity (Fig. 10). To our opinion, the antibiofilm activity of the obtained coatings against the two tested microbial strains could be assigned to the difference between the structure and electric charge of bacterial wall, mediating the interaction with the obtained coatings.
4. Conclusions The aim of this study was to study the possibility to fabricate functional gradient coatings consisting of two biopolymers assembled onto a single substrate in a single step. C-MAPLE technique proved adequate for such a challenge and lead to the fabrication of a gradient thin film from two distinct materials. Indeed, FTIR spectroscopy investigations demonstrated that both SHL and QCH biopolymers preserved the composition of the starting materials after laser transfer and assembling as thin films by C-MAPLE. SEM analyses evidenced that the obtained coatings are homogenous but exhibit specific surface features of each biopolymer. AFM measurements confirmed the gradual modification of roughness parameters along the combinatorial coatings. The proliferation assays demonstrated that the obtained thin films are biocompatible, with distinct increased response of the L929 fibroblast cells to specific surface region estimated to consist in a mixture of about 75% QCH and 25% SHL. SHL biopolymer coatings showed the highest clotting activity in time. The blood clotting capacity decreases with the onset of QCH biopolymer. The antibiofilm activity assay revealed that
Fig. 9. Temporal dynamics of the S. aureus biofilm growth on the C-MAPLE coatings. 634
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all regions of the coatings are impairing the adhesion of both initial and mature biofilms of E. coli cells. In case of the S. aureus, the biofilm was inhibited only in the initial adherence stage by the samples containing the highest amounts of levan and chitosan, respectively.
102 (2017) 565–570. [20] A.D. Sezer, H. Kazak, E.T. Öner, J. Akbuğa, Levan-based nanocarrier system for peptide and protein drug delivery: optimization and influence of experimental parameters on the nanoparticle characteristics, Carbohydr. Polym. 84 (1) (2011) 358–363. [21] M.S. Bostan, E.C. Mutlu, H. Kazak, S.S. Keskin, E.T. Oner, M.S. Eroglu, Comprehensive characterization of chitosan/PEO/levan ternary blend films, Carbohydr. Polym. 102 (2014) 993–1000. [22] R.R. Costa, A. Girotti, M. Santos, F.J. Arias, J.F. Mano, J.C. Rodríguez-Cabello, Cellular uptake of multilayered capsules produced with natural and genetically engineered biomimetic macromolecules, Acta Biomater. 10 (6) (2014) 2653–2662. [23] A. Osman, E.T. Oner, M.S. Eroglu, Novel levan and pNIPA temperature sensitive hydrogels for 5-ASA controlled release, Carbohydr. Polym. 165 (2017) 61–70. [24] A. Berg, E.T. Oner, J. Combie, B. Schneider, R. Ellinger, J. Weisser, R. Wyrwa, M. Schnabelrauch, Formation of new, cytocompatible hydrogels based on photochemically crosslinkable levan methacrylates, Int. J. Biol. Macromol. (2017). [25] G. Avsar, D. Agirbasli, M.A. Agirbasli, O. Gunduz, E.T. Oner, Levan based fibrous scaffolds electrospun via co-axial and single-needle techniques for tissue engineering applications, Carbohydr. Polym. (2018). [26] T.D. Gomes, S.G. Caridade, M.P. Sousa, S. Azevedo, M.Y. Kandur, E.T. Öner, N.M. Alves, J.F. Mano, Adhesive free-standing multilayer films containing sulfated levan for biomedical applications, Acta Biomater. 69 (2018) 183–195. [27] F. Sima, E.C. Mutlu, M.S. Eroglu, L.E. Sima, N. Serban, C. Ristoscu, S.M. Petrescu, E.T. Oner, I.N. Mihailescu, Levan nanostructured thin films by MAPLE assembling, Biomacromolecules 12 (6) (2011) 2251–2256. [28] J. Zhang, W. Xia, P. Liu, Q. Cheng, T. Tahi, W. Gu, B. Li, Chitosan modification and pharmaceutical/biomedical applications, Mar. Drugs 8 (7) (2010) 1962–1987. [29] P.K. Dutta, J. Dutta, V. Tripathi, Chitin and chitosan: chemistry, properties and applications, J. Sci. Ind. Res. 63 (1) (2004) 20–31. [30] M.H. Periayah, A.S. Halim, A.Z.M. Saad, Chitosan: a promising marine polysaccharide for biomedical research, Pharmacogn. Rev. 10 (19) (2016) 39. [31] D.E.-A.-K.a.M.R. Hamblin, Chitin and chitosan: production and application of versatile biomedical nanomaterials, Int. J. Adv. Res. 4 (3) (2016) 411–427. [32] P. Sandford, Chitosan and alginate-new forms of commercial interest, Abstracts Of Papers Of The American Chemical Society, AMER Chemical Soc 1155 16th St, Nw, Washington, DC 20036 (1990) 298 -POLY. [33] J.R. Chavda, Studies in Design and Development of Stomach Specific Drug Delivery System Using Various Approaches, Saurashtra University, 2010. [34] A. Francesko, T. Tzanov, Chitin, Chitosan and Derivatives for Wound Healing and Tissue Engineering, Biofunctionalization of Polymers and their Applications, Springer, 2010, pp. 1–27. [35] J.D. Bumgardner, R. Wiser, P.D. Gerard, P. Bergin, B. Chestnutt, M. Marini, V. Ramsey, S.H. Elder, J.A. Gilbert, Chitosan: potential use as a bioactive coating for orthopaedic and craniofacial/dental implants, J. Biomater. Sci. Polym. Ed. 14 (5) (2003) 423–438. [36] M.R. Leedy, H.J. Martin, P.A. Norowski, J.A. Jennings, W.O. Haggard, J.D. Bumgardner, Use of chitosan as a bioactive implant coating for bone-implant applications, Chitosan for Biomaterials II, Springer, 2011, pp. 129–165. [37] N. Poth, V. Seiffart, G. Gross, H. Menzel, W. Dempwolf, Biodegradable chitosan nanoparticle coatings on titanium for the delivery of BMP-2, Biomolecules 5 (1) (2015) 3–19. [38] S.K. Mishra, J. Ferreira, S. Kannan, Mechanically stable antimicrobial chitosan–PVA–silver nanocomposite coatings deposited on titanium implants, Carbohydr. Polym. 121 (2015) 37–48. [39] A. Bernkop-Schnürch, S. Dünnhaupt, Chitosan-based drug delivery systems, Eur. J. Pharm. Biopharm. 81 (3) (2012) 463–469. [40] M.S. Bostan, M. Senol, T. Cig, I. Peker, A.C. Goren, T. Ozturk, M.S. Eroglu, Controlled release of 5-aminosalicylicacid from chitosan based pH and temperature sensitive hydrogels, Int. J. Biol. Macromol. 52 (2013) 177–183. [41] Y. Sun, F. Cui, K. Shi, J. Wang, M. Niu, R. Ma, The effect of chitosan molecular weight on the characteristics of spray-dried methotrexate-loaded chitosan microspheres for nasal administration, Drug Dev. Ind. Pharm. 35 (3) (2009) 379–386. [42] J. Kumirska, M.X. Weinhold, J. Thöming, P. Stepnowski, Biomedical activity of chitin/chitosan based materials—influence of physicochemical properties apart from molecular weight and degree of N-acetylation, Polymers 3 (4) (2011) 1875–1901. [43] Z.I. Al-Kurdi, B.Z. Chowdhry, S.A. Leharne, M.M. Al Omari, A.A. Badwan, Low molecular weight chitosan-insulin polyelectrolyte complex: characterization and stability studies, Mar Drugs 13 (4) (2015) 1765–1784. [44] M.N. Ravi Kumar, A review of chitin and chitosan applications, Reactive Funct. Polym. 46 (1) (2000) 1–27. [45] M. Lavertu, S. Methot, N. Tran-Khanh, M.D. Buschmann, High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation, Biomaterials 27 (27) (2006) 4815–4824. [46] G. Cravotto, S. Tagliapietra, B. Robaldo, M. Trotta, Chemical modification of chitosan under high-intensity ultrasound, Ultrason. Sonochem. 12 (1) (2005) 95–98. [47] E. Tang, M. Huang, L. Lim, Ultrasonication of chitosan and chitosan nanoparticles, Int. J. Pharm. 265 (1) (2003) 103–114. [48] H.K. No, J.W. Nah, S.P. Meyers, Effect of time/temperature treatment parameters on depolymerization of chitosan, J. Appl. Polym. Sci. 87 (12) (2003) 1890–1894. [49] S. Mao, X. Shuai, F. Unger, M. Simon, D. Bi, T. Kissel, The depolymerization of chitosan: effects on physicochemical and biological properties, Int. J. Pharm. 281 (1) (2004) 45–54. [50] M. Agirre, J. Zarate, E. Ojeda, G. Puras, J. Desbrieres, J.L. Pedraz, Low molecular weight chitosan (LMWC)-based polyplexes for pDNA delivery: from bench to bedside, Polymers 6 (6) (2014) 1727–1755.
Acknowledgments NM acknowledges with thanks the support of this work by UEFISCDI under the PD_52/2018 contract. MS, CR and INM acknowledge NATO and UEFISCDI for the support of this work under the contracts G4890 and 43NATO/2017. MS and EA acknowledge with thanks the support of this work by UEFISCDI under the 131PED/2017 contract and Nucleus programme – LAPLAS V. MSE and MSB gratefully acknowledge “Marmara University Research Fund (BABKO) Project Number FEN-C-DRP-031210-0290”. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsusc.2018.10.077. References [1] M. Håkanson, E. Cukierman, M. Charnley, Miniaturized pre-clinical cancer models as research and diagnostic tools, Adv. Drug Delivery Rev. 69 (2014) 52–66. [2] R. Liu, X. Li, W. Xiao, K.S. Lam, Tumor-targeting peptides from combinatorial libraries, Adv. Drug Deliv. Rev. 110 (2017) 13–37. [3] O.Z. Fisher, A. Khademhosseini, R. Langer, N.A. Peppas, Bioinspired materials for controlling stem cell fate, Acc. Chem. Res. 43 (3) (2009) 419–428. [4] F. Sima, E. Axente, L. Sima, U. Tuyel, M. Eroglu, N. Serban, C. Ristoscu, S. Petrescu, E. Toksoy Oner, I. Mihailescu, Combinatorial matrix-assisted pulsed laser evaporation: single-step synthesis of biopolymer compositional gradient thin film assemblies, Appl. Phys. Lett. 101 (23) (2012) 233705. [5] E. Axente, F. Sima, L.E. Sima, M. Erginer, M.S. Eroglu, N. Serban, C. Ristoscu, S.M. Petrescu, E.T. Oner, I.N. Mihailescu, Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts, Biofabrication 6 (3) (2014) 035010. [6] E. Axente, M. Sopronyi, C.M. Ghimbeu, C. Nita, A. Airoudj, G. Schrodj, F. Sima, Matrix-assisted pulsed laser evaporation: a novel approach to design mesoporous carbon films, Carbon 122 (2017) 484–495. [7] E. Boanini, P. Torricelli, F. Sima, E. Axente, M. Fini, I.N. Mihailescu, A. Bigi, Gradient coatings of strontium hydroxyapatite/zinc β-tricalcium phosphate as a tool to modulate osteoblast/osteoclast response, J. Inorg. Biochem. 183 (2018) 1–8. [8] P. Torricelli, F. Sima, E. Axente, M. Fini, I.N. Mihailescu, A. Bigi, Strontium and zoledronate hydroxyapatites graded composite coatings for bone prostheses, J. Colloid Interface Sci. 448 (2015) 1–7. [9] E. Axente, F. Sima, C. Ristoscu, N. Mihailescu, I.N. Mihailescu, Biopolymer thin films synthesized by advanced pulsed laser techniques, Recent Adv. Biopolym. (2016) InTech. [10] F. Sima, E. Axente, C. Ristoscu, O. Gallet, K. Anselme, I. Mihailescu, Bioresponsive surfaces and interfaces fabricated by innovative laser approaches, Adv. Mater. Interfaces (2016) 427–462. [11] S. Sudharsan, N. Subhapradha, P. Seedevi, V. Shanmugam, P. Madeswaran, A. Shanmugam, A. Srinivasan, Antioxidant and anticoagulant activity of sulfated polysaccharide from Gracilaria debilis Forsskal, Int. J. Biol. Macromol. 81 (2015) 1031–1038. [12] S. li Xiong, A. Li, N. Huang, F. Lu, D. Hou, Antioxidant and immunoregulatory activity of different polysaccharide fractions from tuber of ophiopogon japonicus, Carbohydr. Polym. 86 (3) (2011) 1273–1280. [13] X.-X. Liu, Z.-J. Wan, L. Shi, X.-X. Lu, Preparation and antiherpetic activities of chemically modified polysaccharides from Polygonatum cyrtonema Hua, Carbohydr. Polym. 83 (2) (2011) 737–742. [14] M. Erginer, A. Akcay, B. Coskunkan, T. Morova, D. Rende, S. Bucak, N. Baysal, R. Ozisik, M.S. Eroglu, M. Agirbasli, Sulfated levan from halomonas smyrnensis as a bioactive, heparin-mimetic glycan for cardiac tissue engineering applications, Carbohydr. Polym. 149 (2016) 289–296. [15] V.H. Pomin, P.A. Mourão, Specific sulfation and glycosylation—a structural combination for the anticoagulation of marine carbohydrates, Front. Cell. Infect. Microbiol. 4 (2014). [16] E.T. Öner, L. Hernández, J. Combie, Review of levan polysaccharide: from a century of past experiences to future prospects, Biotechnol. Adv. 34 (5) (2016) 827–844. [17] H.K. Sarilmiser, O. Ates, G. Ozdemir, K.Y. Arga, E.T. Oner, Effective stimulating factors for microbial levan production by Halomonas Smyrnensis AAD6 T, J. Biosci. Bioeng. 119 (4) (2015) 455–463. [18] H.K. Sarilmiser, E.T. Oner, Investigation of anti-cancer activity of linear and aldehyde-activated levan from Halomonas smyrnensis AAD6 T, Biochem. Eng. J. 92 (2014) 28–34. [19] E.A. Queiroz, Z.B. Fortes, M.A. da Cunha, H.K. Sarilmiser, A.M.B. Dekker, E.T. Öner, R.F. Dekker, N. Khaper, Levan promotes antiproliferative and pro-apoptotic effects in MCF-7 breast cancer cells mediated by oxidative stress, Int. J. Biol. Macromol.
635
Applied Surface Science 466 (2019) 628–636
N. Mihailescu et al.
[60] A.M. Sajjan, H. Premakshi, M.Y. Kariduraganavar, Synthesis and characterization of GTMAC grafted chitosan membranes for the dehydration of low water content isopropanol by pervaporation, J. Ind. Eng. Chem. (2014). [61] M.S. Bostan, Preparation and Characterization of Gene and Drug Delivery Systems Chemical Engineering Marmara University, İstanbul (2015) 145. [62] A. Poli, H. Kazak, B. Gürleyendağ, G. Tommonaro, G. Pieretti, E.T. Öner, B. Nicolaus, High level synthesis of levan by a novel Halomonas species growing on defined media, Carbohydr. Polym. 78 (4) (2009) 651–657. [63] A.F.D. Vasconcelos, R.F. Dekker, A.M. Barbosa, E.R. Carbonero, J.L. Silveira, B. Glauser, M.S. Pereira, M.d.L.C. da Silva, Sulfonation and anticoagulant activity of fungal exocellular β-(1→ 6)-D-glucan (lasiodiplodan), Carbohydr. Polym. 92 (2) (2013) 1908–1914. [64] I.N. Mihailescu, C. Ristoscu, A. Bigi, I. Mayer, Advanced Biomimetic Implants Based on Nanostructured Coatings Synthesized by Pulsed Laser Technologies, in: Antonio Miotello, Paolo M. Ossi (Eds.), Laser-Surface Interactions for New Materials Production Tailoring Structure and Properties Series: Springer Series in Materials Science, 130 2010, pp. 235–260. [65] Stefania Di Cio, Julien E. Gautrot, Cell sensing of physical properties at the nanoscale: mechanisms and control of cell adhesion and phenotype, Acta Biomater. 30 (2016) 26–48. [66] M. Vandrovcová, L. Bačáková, Adhesion, growth and differentiation of osteoblasts on surface-modified materials developed for bone implants, Physiol. Res. 60 (2011) 403–417. [67] H. Li, G. Zhang, J. Wu, C. Zhao, Y. Zhang, K. Shao, M. Han, H. Lin, J. Zhu, H. Na, A novel sulfonated poly (ether ether ketone) and cross-linked membranes for fuel cells, J. Power Sources 195 (19) (2010) 6443–6449. [68] L. Bacakova, E. Filova, F. Rypacek, V. Svorcik, V. Stary, Cell adhesion on artificial materials for tissue engineering, Physiol. Res. 53 (2004) S35–S45.
[51] V. Bansal, P.K. Sharma, N. Sharma, O.P. Pal, R. Malviya, Applications of chitosan and chitosan derivatives in drug delivery, Adv. Biol. Res. 5 (1) (2011) 28–37. [52] R. Riva, H. Ragelle, A. des Rieux, N. Duhem, C. Jérôme, V. Préat, Chitosan and chitosan derivatives in drug delivery and tissue engineering, Chitosan for Biomaterials II, Springer, 2011, pp. 19–44. [53] R.C. Goy, S.T. Morais, O.B. Assis, Evaluation of the antimicrobial activity of chitosan and its quaternized derivative on E. coli and S. aureus growth, Revista Brasileira de, Farmacognosia 26 (1) (2016) 122–127. [54] M. Ignatova, K. Starbova, N. Markova, N. Manolova, I. Rashkov, Electrospun nanofibre mats with antibacterial properties from quaternised chitosan and poly (vinyl alcohol), Carbohydr. Res. 341 (12) (2006) 2098–2107. [55] A.F. Martins, S.P. Facchi, H.D. Follmann, A.G. Pereira, A.F. Rubira, E.C. Muniz, Antimicrobial activity of chitosan derivatives containing N-quaternized moieties in its backbone: a review, Int. J. Mol. Sci. 15 (11) (2014) 20800–20832. [56] L. Cao, H. Zhang, C. Cao, J. Zhang, F. Li, Q. Huang, Quaternized chitosan-capped mesoporous silica nanoparticles as nanocarriers for controlled pesticide release, Nanomaterials 6 (7) (2016) 126. [57] F. Miao, Z. Guo, Y. Yi, S. Yang, The moisture absorption and retention abilities of quaternized carboxymethyl chitosan, Bioinformatics and Biomedical Engineering, 2009 ICBBE 2009. 3rd International Conference on, IEEE, 2009, pp. 1–3. [58] A. Visan, G.E. Stan, C. Ristoscu, G. Popescu-Pelin, M. Sopronyi, C. Besleaga, C. Luculescu, M.C. Chifiriuc, M.D. Hussien, O. Marsan, E. Kergourlay, D. Grossin, F. Brouillet, I.N. Mihailescu, Combinatorial MAPLE deposition of antimicrobial orthopedic maps fabricated from chitosan and biomimetic apatite powders, Int. J. Pharm. 511 (1) (2016) 505–515. [59] R. Deveikytė, R. Makuška, Quaternization of chitosan and partial destruction of the quaternized derivatives making them suitable for electrospinning, Chemija 24 (4) (2013) 325–334.
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Full Length Article
Gradient multifunctional biopolymer thin film assemblies synthesized by combinatorial MAPLE
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Natalia Mihailescua, Merve Erginer Haskoylub, Carmen Ristoscua, Müge Sennaroglu Bostanc, Mihai Sopronyia,d, Mehmet S. Eroğluc,e, Mariana Carmen Chifiriucf, ⁎ Cosmin Catalin Mustaciosug,h, Emanuel Axentea, Ebru Toksoy Onerb, Ion N. Mihailescua, a
National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, Magurele, Ilfov RO-77125, Romania Department of Bioengineering, Marmara University, Goztepe, 34722 Istanbul, Turkey c Department of Chemical Engineering, Marmara University, Goztepe, 34722 Istanbul, Turkey d Physics Department, University of Bucharest, Magurele, Ilfov RO-077125, Romania e TUBITAK-UME, Chemistry Group Laboratories, 41471 Gebze, Kocaeli, Turkey f Department of Microbiology, Faculty of Biology and Research Institute of the University of Bucharest (ICUB), University of Bucharest, 060101 Bucharest, Romania g Horia Hulubei National Institute for Physics and Nuclear Engineering IFIN-HH, Magurele, Ilfov RO-077125, Romania h Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Romania b
A R T I C LE I N FO
A B S T R A C T
Keywords: Combinatorial-MAPLE Biopolymer coatings Compositional maps Sulfated Halomonas Levan - quaternized low molecular weight Chitosan mixtures Anti-biofilm activity
Combinatorial Matrix-Assisted Pulsed Laser Evaporation (C-MAPLE) was recently introduced to the fast generation of compositional libraries of two biopolymers in a single-step process, for tissue engineering and regenerative medicine applications. Synchronized laser irradiation of two distinct cryogenic targets, one consisting of Sulfated Halomonas Levan and the other of quaternized low molecular weight Chitosan was used to fabricate compositional gradient coatings for surface functionalization. Synthesized coatings preserved the base material composition as confirmed by Fourier Transform Infrared Spectroscopy. Morphological study by Scanning Electron Microscopy, Atomic Force Microscopy and profilometry correlated with water contact angles measurements demonstrated that the obtained thin coatings have improved surface properties with respect to pure material coatings. Fluorescence microscopy validated the compositional gradient, while in vitro assays evidenced characteristic responses of mouse fibroblasts (L929 cell line) by distinct deposition surface regions. The coagulation test pointed out good properties for Sulfated Halomonas Levan coatings as compared to the case of an increased amount of quaternized low molecular weight Chitosan biopolymer or the control. The antimicrobial effect of the coatings was demonstrated against Escherichia coli and Staphylococcus aureus strains, representative for both Gram negative and Gram positive bacterial species, respectively, mainly involved in implant and nosocomial infections. The assembled nanostructures possess variable anti-biofilm activity along the compositional gradient, with a stronger inhibitory effect on the initial adherence phase of both tested microbial strains, but also against mature Escherichia coli biofilms. It was shown that C-MAPLE can generate discrete areas of blended polymeric composition exhibiting improved surface properties for a broad range of biomedicine applications, e.g. the fabrication of thin bioactive and cell-instructive coatings with anti-adherence properties.
1. Introduction Recently, functional coatings attracted a rapidly growing interest, due to the possibility to explore novel properties of materials at either micro- or nanoscale. With respect to their original protective function, thin films are nowadays in the forefront of sustained advances in
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fabricating antimicrobial, bioactive and biomimetic, transparent and conductive, thermo-electric, self-healing, self-cleaning and super-hydrophobic coatings, to mention a few applications only. On the other hand, combinatorial research, in its whole spectrum of applications in materials science and chemistry, has proven to drive fast progress in the synthesis of innovative materials or new properties for nanomedicine.
Corresponding author. E-mail address: ion.mihailescu@inflpr.ro (I.N. Mihailescu).
https://doi.org/10.1016/j.apsusc.2018.10.077 Received 7 June 2018; Received in revised form 5 October 2018; Accepted 8 October 2018 Available online 09 October 2018 0169-4332/ © 2018 Elsevier B.V. All rights reserved.
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In the biomedical field, most of the applications are related to the fabrication of bioresponsive surfaces and interfaces able to modulate cells behavior and push forward drug discovery and delivery systems [1,2]. Indeed, combinatorial techniques, correlated with highthroughput screening stand for the best tools to improve the composition-structure and/or properties relationship for both organic and inorganic library coatings [3]. We have recently introduced a single-step combinatorial approach based on Matrix-Assisted Pulsed Laser Evaporation (MAPLE) for both the immobilization and blending of organic and environmental friendly polymeric compounds [4–8]. Later, both experimental and theoretical aspects of C-MAPLE technique were extensively addressed in our studies of different organic, inorganic or composite materials [9,10]. Laser-based synthesis of biopolymers combinatorial coatings is reviewed in Ref. [9]. Chemically modified or natural polysaccharides with sulfate groups have efficient biological characteristics, such as antioxidant and immunomodulation [11,12], antiherpetic [13], antithrombotic and anticoagulant [14,15] action. Levan is a β-2, 6-linked fructose homopolymer synthesized extracellularly from sucrose substrates by various microorganisms. Due to its unique features, it is considered a quite valuable biomaterial with many potential applications in medical, food, pharmaceutical, cosmetics and chemical industries [16]. The Gram negative aerobic bacterial strain Halomonas smyrnensis AAD6T was reported as the first halophilic levan producer [17], besides its antioxidant and anticancer activities [18,19]. Halomonas Levan (HL) was proved suitable for use as drug carrier systems [20], bioactive thin film blends [21], multilayer adhesive films [22], but also temperature responsive [23] and cytocompatible hydrogels [24]. Recently, a sulfated derivative of HL (SHL) was reported as a heparin mimetic anticoagulant [4,14]. Also, electrospun matrices of SHL with anticoagulant activity were found to have high potential to be used in decreasing neointimal proliferation and thrombogenicity of grafts and prosthesis [25]. SHL was shown to improve the mechanical and adhesive properties of multilayered free-standing films and allow myogenic differentiation and lead to cytocompatible and myoconductive films for cardiac tissue engineering applications [26]. Recent studies reported the characteristics of HL and oxidized HL (OHL) thin films synthesized by MAPLE [27]. HL and OHL were combined and binary gradient films were fabricated using the Combinatorial MAPLE (C-MAPLE) technique. In vitro cell culture studies with the SaOS2 bone cell line have shown that the osteoblasts’ extracellular signal-regulated kinase signaling was modulated with different propensity. It was suggested that C-MAPLE could indeed serve as a suitable method for the fabrication of new bioactive surfaces controlling the cell response [5]. Chitosan (CH) is the deacetylated product of the well-known natural biopolymer chitin. Its chemical reactivity is attractive due to the presence of –NH2 groups [28]. Chitin and Chitosan are highly basic unlike most other natural polysaccharides [29]. Chemical properties of CH include the linear polyamine structure, reactive amino and hydroxyl groups and possibility to chelate transitional metal ions. CH is a natural, biocompatible, biodegradable, safe, non-toxic polymer [30,31]. It has hemostatic, anti-thrombogenic, antibacterial, fungistatic, spermicidal, immunoadjuvant, antitumor, anti-cholesteremic biological properties and may accelerate bone formation and so can be used as central nervous system depressant [32,33]. Because of diverse bioactivities, CH can be applied in a wide range of biomedical applications such as wound healing or tissue engineering [31,34], implant coatings [35–38] and drug delivery systems [28,31,39,40]. CH molecular weight exerts a major influence upon its biological and physical-chemical properties. Thus, the crystallinity, degradation, tensile strengths and moisture content have been shown to be dependent on the molecular weight [30,41–44]. As known, commercially available CH has a high molecular weight. Accordingly, methods have been developed using ultrasound, heat, enzymatic or chemical
hydrolysis to depolymerize CH. Depolymerization of CH using nitrous acid (HNO2) was imposing as a preferred technique, because it is rapid, cost-effective and can be monitored to produce CH of a pre-selected size [45–49]. The way to improve or add new properties to CH [50] is to chemically modify the chain by adding functional groups to the main one, non-altering the original structure and properties. The primary amine and hydroxyl groups (OH) are responsible for reactions such as e.g. quaternization. Moreover, the presence of these reactive primary amino groups adds special properties to CH which make them very appropriate in pharmaceutical applications [51–53]. There are several studies in literature on CH quaternization, reporting the improvement, even under basic conditions, of properties such as the antimicrobial activity [53–56], moisture-retention capacity [57,58], bio-adhesivity, permeation enhancing effect and antifungal activity [56,58]. The possibility to deposit two natural, biocompatible, non-toxic polysaccharides, i.e. Sulfated Halomonas Levan (SHL) and quaternized highly deacetylated low molecular weight CH (QCH) on Si, glass or Ti substrates is explored in this study. We mainly aimed to fabricate thin coatings able to offer a suitable interface for cells leading to an improved osseointegration and simultaneously exhibiting good antimicrobial properties. To this purpose, C-MAPLE method was applied to transfer and deposit SHL and QCH in a single process in order to produce new organic thin libraries with improved properties. To the best of our knowledge, this is a first attempt to synthesize in situ SHL and QCH thin coatings for the selection of best bioactive surface able to control cells behavior and ensure protection against microbial colonization and biofilms formation. 2. Materials and methods 2.1. Synthesis of combinatorial functional coatings by C-MAPLE The preparation of quaternized highly deacetylated and low molecular weight CH (QCH) was carried out via established protocols described in literature [21,40,49,58–60] (and given in detail in Supplementary Information (SI) section). The sulfonation of Halomonas Levan (SHL) was achieved following the procedure described in Refs. [14,25,26] (also detailed in SI). First, 25 mg of SHL were dissolved in 5 mL of dimethyl sulfoxide (DMSO) to obtain a homogeneous solution. DMSO was selected as solvent because it does not chemically interact with SHL, and efficiently absorbs the laser wavelength (248 nm) used for the evaporation of the target in frozen state [27]. A concentration of 2% QCH dissolved in deionized H2O (dH2O), was used for the preparation of the second CMAPLE target. The two solutions were transferred into distinct compartments of a ring-like concentric copper holder. This holder has been designed and manufactured to accommodate in each compartment between 5 and 10 mL of solution to be used in C–MAPLE experiments. The holder containing the solutions was carefully immersed in liquid nitrogen (LN) to get the two cryogenic targets. Next, the holder was mounted inside the cooler, which was under the continuous supply of LN, to keep the target frozen. The schematic representation of the C-MAPLE experimental set-up is depicted in Fig. 1. The laser beam generated by a KrF* excimer laser source (λ = 248 nm, τFWHM = 25 ns), operating at a repetition rate of 10 Hz is divided by an optical splitter. The two beams are directed and focused onto the two concentric targets. The distance between the centers of the two laser spots is set at 2 cm. The evaporated polymers were collected either onto glass, Si (1 0 0) or Ti substrates placed parallel to the targets, at a separation distance of 5.5 cm. Before deposition, all substrates were successively cleaned in an ultrasonic bath for 15 min in acetone, alcohol and dH2O. For the growth of each SHL to QCH coatings, 40.000 laser pulses have been applied. One obtains in this configuration the polymer co-deposition onto a 4 cm long sample, the edges containing 100% SHL at one end and 100% QCH at the other one. A continuous gradient of SHL–QCH blended composition is reached in 629
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by using an UV lamp in a biological cabinet for one hour. 2.3.1. Viability/MTS assay CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay – MTS (Promega) was used for the viability evaluation of L929 mouse fibroblasts (cell line between passages 11 and 14). The cells were detached with trypsin/EDTA (Biochrom) and resuspended in complete medium – MEM with 10% fetal bovine serum (FBS) and L-glutamine at a density of 200,000 cells per mL. Cells were cultured for 30 min in an incubator, so that to firmly adhere to coatings surface. After 24 h incubation, the coatings were moved in new wells. 400 µL of MTS solution were added in MEM with 5% serum and cells were incubated again for 3 h. 100 µL of medium from each well was poured in a 96-well plate, for an absorbance measurement at 490 nm. 2.3.2. Cell morphology examination Cells morphology was assessed using fluorescence microscopy imaging (Olympus BX51fluorescence microscope). The cells were fixed after 24 h incubation using 4% paraformaldehyde, permeated with 0.1% Triton X and incubated overnight at 4 °C with Fluorescein Phalloidin (Thermo Fisher). Hoechst stain (Thermo Fisher) was added for nuclei visualization and the samples were mounted on the slide with ProLongGlodantifade reagent (Thermo Fisher).
Fig. 1. C-MAPLE experimental setup.
between [5,7,8].
2.3.3. Coagulation test Coagulation test (clotting assay on microscope slide) was used for evaluation of the anticoagulant properties of deposited coatings as compared with controls (glass slide). Venous blood from three clinically healthy men, aged 25, 30 and 33 was collected and mixed in equal proportions with Phosphate Buffered Saline (PBS), homogenized and deposited into droplets on coated surfaces. The coatings were previously placed in a Petri dish on a bed of wet tissue to avoid drying. The plates were inclined slightly from time to time to observe the coagulation of the blood with the formation of clot. When the drop stopped moving it was considered that the blood coagulated. Coagulation experiments were performed at RT.
2.2. Physical–chemical characterization of thin gradient coatings The chemical composition of the thin films libraries was studied by Fourier Transform Infrared (FTIR) Spectroscopy with a Shimadzu 8400S FTIR apparatus. Spectra were recorded within the range 4000–500 cm−1, with a resolution of 8.0 cm−1 and a total of 80 scans/ experiment. The measurements were performed along the longitudinal direction of the thin gradient coatings (x direction in Fig. 1). Scanning Electron Microscopy (SEM) was used to examine the surface morphology of the deposited coatings with a FEI Inspect S electron microscope. The measurements were carried out at 20 kV acceleration voltages, in high vacuum, under secondary electrons acquisition mode. Before SEM, the samples were coated with a very thin layer of gold. Atomic Force Microscopy (AFM) with a Workshop TT-AFM apparatus was performed for surface roughness investigations. Images were acquired from different areas of each coating in non-contact mode. Profilometry analysis was done for thickness investigations with an Ambios XP-2 apparatus for a measurement length of 1.5 mm, weight 5 mg, speed 0.01 mm/sec. A step between substrate and thin films was imprinted on each sample and the measurements were carried out on three distinct points for each coating. For statistics, measurements were carried out on three identical samples. SHL and QCH optical and fluorescence microscopy images were acquired with a DM 4000B LED, Leica microscope. Twin images were captured along the C-MAPLE generated SHL–QCH gradient keeping the same exposure time for detection of fluorescence emission between specimens. Contact Angle (CA) measurements by sessile drop technique in static mode were conducted to inspect the hydrophobic/hydrophilic features of the coatings. A Krüss DSA-100 model CA meter equipped with High Performance Frame Grabber Camera T1C (25 frames per second) was used with DS3210 software controlled auto-dosing system. Drops of 5 µL of dH2O were deposited on a dry and clean surface using the autodosing system. Static measurements were conducted immediately after the sessile drop formation and at least five measurements were performed on each sample, at ambient temperature and humidity. The results were statistically averaged.
2.3.4. Anti-biofilm activity assay The anti-biofilm activity of the obtained coatings was carried out with Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8739 strains. The tested coatings were previously sterilized by exposure to UV for 30 min. After UV sterilization, an amount of 20 µL of microbial suspension of 0.5 MacFarland density prepared from fresh cultures developed on tryptic soy agar (TSA) were distributed over the coatings treated surface and incubated at 37 °C in humid atmosphere for 8 or 24 h. Immediately after deposition and after each incubation period, the coatings were suspended in 5 mL sterile saline, vortexed vigorously to resuspend the adherent bacteria. Serial ten-fold dilutions were performed from the recovered suspension, spotted on solid medium in triplicate spots of 10 µL each and the viable cells counts were determined and expressed as colony forming units (CFU)/mL. 3. Results and discussion 3.1. Pure biopolymers FTIR spectrum of QCH (Fig. 2) revealed the presence of all characteristic bands. This is supported by the broad eOH and sharp NeH absorption peaks of CH at 3400 and 3100 cm−1, and CeH bond in eCH2 (ʋ1 = 2920 cm−1) and eCH3 (ʋ2 = 2875 cm−1) groups, respectively [58]. Peaks observed in the range of 1200–1000 cm−1 are assigned to the CeOeC bending vibration of glucose rings and glucose amine bonds, proving that the main chain of CH was not decomposed by oxidation and quaternization. The peak at 1548 cm−1 is assigned to the NeH bending vibration of glucose amine groups. The peaks at 1641 and 681 cm−1 belong to the NeC bending vibration of
2.3. Biological tests Prior to in vitro assay, all coatings and glass controls were sterilized 630
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Fig. 3. FTIR Spectra along the thin film from Levan (SHL) to Chitosan (QCH).
Fig. 2. FTIR spectrum of QCH powder.
3.2.2. SEM and profilometry The surface morphology of SHL, SHL/QCH, and QCH/SHL and QCH coatings is visible in Fig. 4. The coatings consist of a homogenous structure along longitudinal axis of the glass slide from SHL to QCH regions, as revealed by higher magnification in Fig. 4 bottom row. One may notice the display of feature characteristics to each compound. However, at macroscale (Fig. 4 top row), an irregular but interconnected polymer network of quasi-spherical particles is evidenced in the case of SHL, which decrease with QCH content in the combinatorial samples. The profilometry measurements converged to thickness mean values of: ∼450 nm with a mean Standard Deviation (SD) of ± 0.37 SD for SHL, ∼320 nm ± 0.17 SD for SHL/QCH, ∼350 nm ± 0.21 SD for QCH/SHL, and ∼200 nm ± 0.13 SD for QCH coatings. One can estimate on this basis the following growth rate for each coating of: SHL = 0.007; SHL/QCH = 0.011; QCH/SHL = 0.009 and QCH = 0.005 Å/laser pulse.
glycidyltrimethyl ammonium chloride (GTMAC), and stand for a clear evidence of CH quaternization [59]. Additionally, the band visible at around 1475 cm−1 is attributed to the eCH3 groups of GTMAC as confirmed by the study in Ref. [60]. Deacetylation degree was taken as 98% by 1H NMR spectroscopy, in agreement with Refs. [21,40,61], while the conversion of quaternization reaction was found as 28% of the glucosamine rings (see Fig. S2 in SI for details). Molar mass values of CH, DCH and QCH were estimated by online Gel permeation chromatography (as described in SI) and collected in Table 1.
3.2. Characterization of deposited coatings 3.2.1. FTIR Composition was inferred from infrared absorption spectra (Fig. 3) recorded for four representative zones from SHL to QCH thin C-MAPLE coatings. One mentions that the absorption band of HL at 3350 cm−1 is characteristic to the eOH stretching of fructofuranose rings and eCH2 OH groups, respectively. CeH stretching vibration of fructose residues group is supported by the appearance of the two peaks at around 2920 and 2873 cm−1. The weak absorption peak visible at ∼930 cm−1, together with the sharp bands at 1040 and 1130 cm−1 are assigned to the CeOeC symmetric bending vibration of fructofuranose rings and glycosidic linkages [62]. The absorption bands peaking around 1203 and 830 cm−1 are attributed to SHL and support the asymmetric stretching vibration of S]O linkages and symmetrical CeOeS vibration of CeOeSO3 groups, respectively [14,63]. The intensity decrease of all specific absorption bands of SHL is visible and assigned to QCH blending. The major absorption bands of QCH, occurring at 1646, 1588 and 1324 cm−1, are characteristic to eC]O vibration of Amide I, NeH bending, eNH2 to Amide II, and NeH bending to Amide III, respectively. The CeOeC bending vibration of glucose rings and glycosidic linkages appear at 1076, 1021 and 972 cm−1. FTIR investigation convincingly demonstrated the gradient composition from SHL to QCH obtained by C–MAPLE.
3.2.3. AFM High resolution topographic images for SHL to QCH coatings deposited by C-MAPLE were acquired by AFM (Fig. 5). Image analyses revealed that SHL coating exhibits the following roughness parameters: Ra = 11.2 nm and Rms = 14 nm. Nanoparticles evenly spread out over the entire scanned area have dimensions ranging from below 100 nm to 800 nm. The SHL/QCH image is characterized by roughness parameters of Ra = 23.8 nm and Rms = 38.5 nm. An increased magnification indicates the presence of QCH nanoparticles with dimensions of ∼100 nm, which are well visible. They are closely packed with a tendency to form a continuous coating. The largest particles with diameter of about 600 nm are placed near the center of the coating. They are the most probably clusters of material forming during deposition. In case of QCH/SHL coating the roughness parameters increase to Ra = 101 nm and Rms = 140 nm exhibiting higher magnification details (∼400 nm) as compared to SHL and SHL/QCH regions. Nanoparticles with a dimension within the 50 ÷ 500 nm range are more compactly packed in case of QCH and QCH/SHL vs. SHL and SHL/QCH coatings, suggesting a clear clusterization tendency (Fig. 5). Islands of nanoparticles start forming, as visible in the center of the image. AFM investigations of simple QCH coating evidence roughness parameters: Ra = 91.2 nm and Rms = 118 nm, exhibiting lower magnification details (∼200 nm) than QCH/SHL coating. Nanoparticles of 100 ÷ 300 nm diameter are visible agglomerated in clusters which are assembling in form of irregular islands with dimension of 2 ÷ 5 µm (the lighter areas) separated by lower magnification areas (the dark ones). The average magnification of the topography is ∼300 nm. By correlating AFM and SEM investigations, one may conclude that
Table 1 Number Average molecular weight (Mn), Weight Average molecular weight (Mw) and polydispersity index (PDI) values for original (CH), deacetylated (DCH) and quaternized (QCH) chitosan. Sample
Mn (g/mol)
Mw (g/mol)
Mw/Mn (PDI)
CH DCH QCH
9.475e+4 (0.4%) 7.319e+4 (0.4%) 5.383e+3 (3%)
1.670e+5 (0.3%) 1.177e+5 (0.3%) 7.023e+3 (3%)
1.763 (0.5%) 1.608 (0.5%) 1.305 (5%)
631
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Fig. 4. SEM micrographs of SHL (left) to QCH (right) gradient coatings prepared by C-MAPLE. The images were collected at two magnifications: top row is at 1000x magnification with a scale bar of 50 µm and bottom row is at 20,000x magnification with a scale bar of 4 µm.
QCH samples exhibit more homogenous surface features in respect with SHL coatings. On the other hand, roughness increases with the content of QCH, which has Rms of 118 nm while this is of only 14 nm for SHL surface. Since SHL has a higher molecular weight (> 2 * 106 g/mol; [25]) than QCH, the observed surface roughness difference between samples could be due to distinct chain lengths and polarities between sulfate and tri-methyl ammonium groups. The higher polarity of sulfate groups of SHL provided better contact with the glass substrate which leads to lower roughness. It should be stressed out that the presence of particulates of various shapes all over the gradient coatings is an intrinsic characteristic of this deposition method. One therefore expects benefices due to the increased surface roughness resulting in a better insitu anchorage of the implant. Micro-movements of the medical device are minimal therefore ensuring the required primary immovability till bone grows and attaches to the implant surface. Moreover, these particulates are beneficial for the growth and proliferation of cells because they favour the better anchorage of cells roots or cytoplasmatic extensions [64]. Recently, it was demonstrated that cells respond to nanoscale physical properties of the biomaterial [65]. The nanoscale topography provides superior support for the proliferation of bone cells due to the advantageous interaction between nanosize irregularities on the material surface and adsorbed cell adhesion-mediating molecules [66]. Accordingly, a surface roughness in tens of nanometers range is desired for the adhesion, growth, differentiation and phenotypic maturation of bone cells, rather than flat surfaces or surfaces with submicron- or micro-scale roughness.
Table 2 CA results. Sample
Contact Angle [˚]
Control -glass slide SHL SHL/QCH QCH/SHL QCH
66.1 13.0 85.2 84.1 66.3
than weak carboxyl groups and absorb more water [67]. QCH region points to almost the same CA value as the control sample (Table 2). When SHL is combined with QCH, as e.g. in the central region of the combinatorial coating, one observes that hydrophobicity of both polymers is increasing. Biopolymers blending and assembling on substrate generate more hydrophobic surface features as compared with native ones. According to AFM data, increased roughness is resulting in a lower wettability of the surface. Concerning SHL/QCH and QCH/SHL structures, as neutralization is reached with the opposite charges along with the increase of the roughness, a higher CA was recorded as compared to native polymers. Previous studies demonstrated that MAPLE layers of Oxidized Halomonas Levan behave more hydrophilic than Halomonas Levan as a result of the acidic aldehyde-hydrogen bonds [27]. SHL showed noticeably higher hydrophilicity (CA = 13°) compared to native Levan (CA = 58°) as reported in Ref. [27], due to the presence of sulfonate groups. Moreover, moderate hydrophylicity/hydrophobicity favours cell-substrate interaction [68].
3.2.4. Contact angle (CA) measurements CA results obtained with dH2O sessile drops on control (uncovered glass slide), SHL, SHL/QCH, QCH/SHL and QCH C–MAPLE deposited coatings are collected in Table 2. Images revealed that SHL coating has a highly hydrophilic surface characterized by a CA of 13.0°, in agreement with previous studies [14]. As known, strong sulfonate groups have shorter water residence times
3.2.5. Fluorescence microscopy The fluorescence signal evolution along the coatings from SHL to QCH was investigated to qualitatively evidence the gradient between the two polysaccharides. Paired optical and fluorescence microscopy images of SHL to QCH vs. glass control, are given in Fig. 6a. As visible, an almost linear evolution of QCH fluorescence intensity signal is
Fig. 5. AFM images of investigated areas of SHL to QCH coatings. The SHL and SHL/QCH images were acquired on 5 × 5 µm2 area, and the QCH/SHL and QCH images were collected from a 15 × 15 µm2 area. 256 × 256 lines and 0.5 Hz scan frequency were used. 632
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Fig. 6. (a) Fluorescence microscopy and optical images of SHL–QCH (up and down, respectively) gradient coatings obtained by C-MAPLE along the glass slide. Glass controls are given. Magnification Bar = 100 µm (20×). (b) Schematic representation of composition gradient.
reached (Fig. 6b), which serves as a clear demonstration of the composition gradient, in agreement with FTIR spectroscopy investigations.
Table 3 L929 cells viability on C-MAPLE samples and control, as obtained in 4 different experiments.
3.3. Biological tests Experiment Experiment Experiment Experiment
3.3.1. Viability/MTS assay L929 cells viability on the obtained combinatorial coatings containing SHL-QCH mixtures is slightly larger than the glass control (Fig. 7), promoting a stimulating effect of C–MAPLE library on both the cell attachment and growth.
1 2 3 4
Control
SHL
SHL/QCH
QCH/SHL
QCH
1.171 1.223 1.229 1.211
1.224 1.218 1.196 1.236
1.26 1.269 1.218 1.303
1.374 1.343 1.352 1.392
1.323 1.353 1.376 1.359
From statistical point of view, after at least 3 experiments, particularly in the case of QCH/SHL mixture, the cells viability is reproducible and it is superior to the control (Table 3). Moreover, the combination of biopolymers has significantly increased cells viability, specifically in case of QCH/SHL mixture. It reached 110% of the most used control in literature, clearly demonstrating the biocompatibility of the gradient coatings obtained by combinatorial MAPLE. 3.3.2. Cell morphology Fluorescence microscopy images of L929 cells seeded on C–MAPLE samples and on control show that the cells express normal morphology, with a polyhedral and elongated shape, exhibiting a well-established cytoskeleton on both control and coatings. This is in agreement with the results of the MTS assays. Moreover, the nuclear morphology (areas marked with white circles in Fig. 8) indicates that the cells are in various stages of division with a normal proliferative behavior. This stands for a good indicator of the biocompatibility of the C–MAPLE coatings.
Fig. 7. L929 cells proliferation on SHL, SHL/QCH, QCH/SHL, QCH C–MAPLE coatings and on glass control.
3.3.3. Coagulation Data in Table 4 show that the QCH coatings alone have no 633
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Fig. 8. Fluorescence microscopy and Bright Field images of L929 cells onto control (glass) and on SHL, SHL/QCH, QCH/SHL, and QCH C–MAPLE coatings (magnification × 40). Actin fibers are marked with green by Fluorescein-Phalloidin, while Nucleus is stained with blue Hoechst. Table 4 Anticoagulant activity of SHL, SHL/QCH, QCH/SHL, and QCH coatings. Sample
Control (glass)
SHL
SHL/QCH
QCH/SHL
QCH
Time (min)
8
106
40
15
8.5
significant effect on the clotting time. They are similar to the glass control. On the other hand, the clotting time increases when increasing SHL concentration in C–MAPLE samples. SHL coatings exhibit the highest clotting activity. 3.3.4. Antibiofilm assay We have investigated the antibiofilm effect of the obtained bioactive polymeric coatings against S. aureus and E. coli strains. They were selected as relevant Gram positive and Gram negative microorganisms, respectively, which are the most frequently involved in the etiology of medical devices associated infections. Anti-adhesion and antibiofilm activity of the obtained coatings were tested in triplicate, immediately after the deposition of the microbial suspension on the surface of the samples, and after 8 h or 24 h of incubation. The tested samples exhibited variable antibiofilm activity, depending on the incubation time, the gradient of the two polymers and the microbial strain. The coatings containing the highest amount of levan (SHL) or chitosan (QCH) proved the most effective anti-adhesion activity, quantified immediately after the contact with S. aureus cells (T0). In exchange, once the S. aureus biofilm starts to grow (T1), the efficiency of the obtained coatings decreases, becoming comparable or even lower than that of the uncoated control sample (Fig. 9). In case of E. coli, the initial adherence of the cells quantified at T0 was inferior in case of all types of polymeric coatings, as compared to
Fig. 10. Temporal dynamics of the E. coli biofilm growth on the obtained coatings.
control. After 8 h of incubation (T1) all tested coatings proved a lower antibiofilm activity than the uncoated control sample. In exchange, at 24 h of incubation (T2), all coatings, and particularly the samples SHL/ QCH and QCH/SHL proved a very intensive antibiofilm activity (Fig. 10). To our opinion, the antibiofilm activity of the obtained coatings against the two tested microbial strains could be assigned to the difference between the structure and electric charge of bacterial wall, mediating the interaction with the obtained coatings.
4. Conclusions The aim of this study was to study the possibility to fabricate functional gradient coatings consisting of two biopolymers assembled onto a single substrate in a single step. C-MAPLE technique proved adequate for such a challenge and lead to the fabrication of a gradient thin film from two distinct materials. Indeed, FTIR spectroscopy investigations demonstrated that both SHL and QCH biopolymers preserved the composition of the starting materials after laser transfer and assembling as thin films by C-MAPLE. SEM analyses evidenced that the obtained coatings are homogenous but exhibit specific surface features of each biopolymer. AFM measurements confirmed the gradual modification of roughness parameters along the combinatorial coatings. The proliferation assays demonstrated that the obtained thin films are biocompatible, with distinct increased response of the L929 fibroblast cells to specific surface region estimated to consist in a mixture of about 75% QCH and 25% SHL. SHL biopolymer coatings showed the highest clotting activity in time. The blood clotting capacity decreases with the onset of QCH biopolymer. The antibiofilm activity assay revealed that
Fig. 9. Temporal dynamics of the S. aureus biofilm growth on the C-MAPLE coatings. 634
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all regions of the coatings are impairing the adhesion of both initial and mature biofilms of E. coli cells. In case of the S. aureus, the biofilm was inhibited only in the initial adherence stage by the samples containing the highest amounts of levan and chitosan, respectively.
102 (2017) 565–570. [20] A.D. Sezer, H. Kazak, E.T. Öner, J. Akbuğa, Levan-based nanocarrier system for peptide and protein drug delivery: optimization and influence of experimental parameters on the nanoparticle characteristics, Carbohydr. Polym. 84 (1) (2011) 358–363. [21] M.S. Bostan, E.C. Mutlu, H. Kazak, S.S. Keskin, E.T. Oner, M.S. Eroglu, Comprehensive characterization of chitosan/PEO/levan ternary blend films, Carbohydr. Polym. 102 (2014) 993–1000. [22] R.R. Costa, A. Girotti, M. Santos, F.J. Arias, J.F. Mano, J.C. Rodríguez-Cabello, Cellular uptake of multilayered capsules produced with natural and genetically engineered biomimetic macromolecules, Acta Biomater. 10 (6) (2014) 2653–2662. [23] A. Osman, E.T. Oner, M.S. Eroglu, Novel levan and pNIPA temperature sensitive hydrogels for 5-ASA controlled release, Carbohydr. Polym. 165 (2017) 61–70. [24] A. Berg, E.T. Oner, J. Combie, B. Schneider, R. Ellinger, J. Weisser, R. Wyrwa, M. Schnabelrauch, Formation of new, cytocompatible hydrogels based on photochemically crosslinkable levan methacrylates, Int. J. Biol. Macromol. (2017). [25] G. Avsar, D. Agirbasli, M.A. Agirbasli, O. Gunduz, E.T. Oner, Levan based fibrous scaffolds electrospun via co-axial and single-needle techniques for tissue engineering applications, Carbohydr. Polym. (2018). [26] T.D. Gomes, S.G. Caridade, M.P. Sousa, S. Azevedo, M.Y. Kandur, E.T. Öner, N.M. Alves, J.F. Mano, Adhesive free-standing multilayer films containing sulfated levan for biomedical applications, Acta Biomater. 69 (2018) 183–195. [27] F. Sima, E.C. Mutlu, M.S. Eroglu, L.E. Sima, N. Serban, C. Ristoscu, S.M. Petrescu, E.T. Oner, I.N. Mihailescu, Levan nanostructured thin films by MAPLE assembling, Biomacromolecules 12 (6) (2011) 2251–2256. [28] J. Zhang, W. Xia, P. Liu, Q. Cheng, T. Tahi, W. Gu, B. Li, Chitosan modification and pharmaceutical/biomedical applications, Mar. Drugs 8 (7) (2010) 1962–1987. [29] P.K. Dutta, J. Dutta, V. Tripathi, Chitin and chitosan: chemistry, properties and applications, J. Sci. Ind. Res. 63 (1) (2004) 20–31. [30] M.H. Periayah, A.S. Halim, A.Z.M. Saad, Chitosan: a promising marine polysaccharide for biomedical research, Pharmacogn. Rev. 10 (19) (2016) 39. [31] D.E.-A.-K.a.M.R. Hamblin, Chitin and chitosan: production and application of versatile biomedical nanomaterials, Int. J. Adv. Res. 4 (3) (2016) 411–427. [32] P. Sandford, Chitosan and alginate-new forms of commercial interest, Abstracts Of Papers Of The American Chemical Society, AMER Chemical Soc 1155 16th St, Nw, Washington, DC 20036 (1990) 298 -POLY. [33] J.R. Chavda, Studies in Design and Development of Stomach Specific Drug Delivery System Using Various Approaches, Saurashtra University, 2010. [34] A. Francesko, T. Tzanov, Chitin, Chitosan and Derivatives for Wound Healing and Tissue Engineering, Biofunctionalization of Polymers and their Applications, Springer, 2010, pp. 1–27. [35] J.D. Bumgardner, R. Wiser, P.D. Gerard, P. Bergin, B. Chestnutt, M. Marini, V. Ramsey, S.H. Elder, J.A. Gilbert, Chitosan: potential use as a bioactive coating for orthopaedic and craniofacial/dental implants, J. Biomater. Sci. Polym. Ed. 14 (5) (2003) 423–438. [36] M.R. Leedy, H.J. Martin, P.A. Norowski, J.A. Jennings, W.O. Haggard, J.D. Bumgardner, Use of chitosan as a bioactive implant coating for bone-implant applications, Chitosan for Biomaterials II, Springer, 2011, pp. 129–165. [37] N. Poth, V. Seiffart, G. Gross, H. Menzel, W. Dempwolf, Biodegradable chitosan nanoparticle coatings on titanium for the delivery of BMP-2, Biomolecules 5 (1) (2015) 3–19. [38] S.K. Mishra, J. Ferreira, S. Kannan, Mechanically stable antimicrobial chitosan–PVA–silver nanocomposite coatings deposited on titanium implants, Carbohydr. Polym. 121 (2015) 37–48. [39] A. Bernkop-Schnürch, S. Dünnhaupt, Chitosan-based drug delivery systems, Eur. J. Pharm. Biopharm. 81 (3) (2012) 463–469. [40] M.S. Bostan, M. Senol, T. Cig, I. Peker, A.C. Goren, T. Ozturk, M.S. Eroglu, Controlled release of 5-aminosalicylicacid from chitosan based pH and temperature sensitive hydrogels, Int. J. Biol. Macromol. 52 (2013) 177–183. [41] Y. Sun, F. Cui, K. Shi, J. Wang, M. Niu, R. Ma, The effect of chitosan molecular weight on the characteristics of spray-dried methotrexate-loaded chitosan microspheres for nasal administration, Drug Dev. Ind. Pharm. 35 (3) (2009) 379–386. [42] J. Kumirska, M.X. Weinhold, J. Thöming, P. Stepnowski, Biomedical activity of chitin/chitosan based materials—influence of physicochemical properties apart from molecular weight and degree of N-acetylation, Polymers 3 (4) (2011) 1875–1901. [43] Z.I. Al-Kurdi, B.Z. Chowdhry, S.A. Leharne, M.M. Al Omari, A.A. Badwan, Low molecular weight chitosan-insulin polyelectrolyte complex: characterization and stability studies, Mar Drugs 13 (4) (2015) 1765–1784. [44] M.N. Ravi Kumar, A review of chitin and chitosan applications, Reactive Funct. Polym. 46 (1) (2000) 1–27. [45] M. Lavertu, S. Methot, N. Tran-Khanh, M.D. Buschmann, High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation, Biomaterials 27 (27) (2006) 4815–4824. [46] G. Cravotto, S. Tagliapietra, B. Robaldo, M. Trotta, Chemical modification of chitosan under high-intensity ultrasound, Ultrason. Sonochem. 12 (1) (2005) 95–98. [47] E. Tang, M. Huang, L. Lim, Ultrasonication of chitosan and chitosan nanoparticles, Int. J. Pharm. 265 (1) (2003) 103–114. [48] H.K. No, J.W. Nah, S.P. Meyers, Effect of time/temperature treatment parameters on depolymerization of chitosan, J. Appl. Polym. Sci. 87 (12) (2003) 1890–1894. [49] S. Mao, X. Shuai, F. Unger, M. Simon, D. Bi, T. Kissel, The depolymerization of chitosan: effects on physicochemical and biological properties, Int. J. Pharm. 281 (1) (2004) 45–54. [50] M. Agirre, J. Zarate, E. Ojeda, G. Puras, J. Desbrieres, J.L. Pedraz, Low molecular weight chitosan (LMWC)-based polyplexes for pDNA delivery: from bench to bedside, Polymers 6 (6) (2014) 1727–1755.
Acknowledgments NM acknowledges with thanks the support of this work by UEFISCDI under the PD_52/2018 contract. MS, CR and INM acknowledge NATO and UEFISCDI for the support of this work under the contracts G4890 and 43NATO/2017. MS and EA acknowledge with thanks the support of this work by UEFISCDI under the 131PED/2017 contract and Nucleus programme – LAPLAS V. MSE and MSB gratefully acknowledge “Marmara University Research Fund (BABKO) Project Number FEN-C-DRP-031210-0290”. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsusc.2018.10.077. References [1] M. Håkanson, E. Cukierman, M. Charnley, Miniaturized pre-clinical cancer models as research and diagnostic tools, Adv. Drug Delivery Rev. 69 (2014) 52–66. [2] R. Liu, X. Li, W. Xiao, K.S. Lam, Tumor-targeting peptides from combinatorial libraries, Adv. Drug Deliv. Rev. 110 (2017) 13–37. [3] O.Z. Fisher, A. Khademhosseini, R. Langer, N.A. Peppas, Bioinspired materials for controlling stem cell fate, Acc. Chem. Res. 43 (3) (2009) 419–428. [4] F. Sima, E. Axente, L. Sima, U. Tuyel, M. Eroglu, N. Serban, C. Ristoscu, S. Petrescu, E. Toksoy Oner, I. Mihailescu, Combinatorial matrix-assisted pulsed laser evaporation: single-step synthesis of biopolymer compositional gradient thin film assemblies, Appl. Phys. Lett. 101 (23) (2012) 233705. [5] E. Axente, F. Sima, L.E. Sima, M. Erginer, M.S. Eroglu, N. Serban, C. Ristoscu, S.M. Petrescu, E.T. Oner, I.N. Mihailescu, Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts, Biofabrication 6 (3) (2014) 035010. [6] E. Axente, M. Sopronyi, C.M. Ghimbeu, C. Nita, A. Airoudj, G. Schrodj, F. Sima, Matrix-assisted pulsed laser evaporation: a novel approach to design mesoporous carbon films, Carbon 122 (2017) 484–495. [7] E. Boanini, P. Torricelli, F. Sima, E. Axente, M. Fini, I.N. Mihailescu, A. Bigi, Gradient coatings of strontium hydroxyapatite/zinc β-tricalcium phosphate as a tool to modulate osteoblast/osteoclast response, J. Inorg. Biochem. 183 (2018) 1–8. [8] P. Torricelli, F. Sima, E. Axente, M. Fini, I.N. Mihailescu, A. Bigi, Strontium and zoledronate hydroxyapatites graded composite coatings for bone prostheses, J. Colloid Interface Sci. 448 (2015) 1–7. [9] E. Axente, F. Sima, C. Ristoscu, N. Mihailescu, I.N. Mihailescu, Biopolymer thin films synthesized by advanced pulsed laser techniques, Recent Adv. Biopolym. (2016) InTech. [10] F. Sima, E. Axente, C. Ristoscu, O. Gallet, K. Anselme, I. Mihailescu, Bioresponsive surfaces and interfaces fabricated by innovative laser approaches, Adv. Mater. Interfaces (2016) 427–462. [11] S. Sudharsan, N. Subhapradha, P. Seedevi, V. Shanmugam, P. Madeswaran, A. Shanmugam, A. Srinivasan, Antioxidant and anticoagulant activity of sulfated polysaccharide from Gracilaria debilis Forsskal, Int. J. Biol. Macromol. 81 (2015) 1031–1038. [12] S. li Xiong, A. Li, N. Huang, F. Lu, D. Hou, Antioxidant and immunoregulatory activity of different polysaccharide fractions from tuber of ophiopogon japonicus, Carbohydr. Polym. 86 (3) (2011) 1273–1280. [13] X.-X. Liu, Z.-J. Wan, L. Shi, X.-X. Lu, Preparation and antiherpetic activities of chemically modified polysaccharides from Polygonatum cyrtonema Hua, Carbohydr. Polym. 83 (2) (2011) 737–742. [14] M. Erginer, A. Akcay, B. Coskunkan, T. Morova, D. Rende, S. Bucak, N. Baysal, R. Ozisik, M.S. Eroglu, M. Agirbasli, Sulfated levan from halomonas smyrnensis as a bioactive, heparin-mimetic glycan for cardiac tissue engineering applications, Carbohydr. Polym. 149 (2016) 289–296. [15] V.H. Pomin, P.A. Mourão, Specific sulfation and glycosylation—a structural combination for the anticoagulation of marine carbohydrates, Front. Cell. Infect. Microbiol. 4 (2014). [16] E.T. Öner, L. Hernández, J. Combie, Review of levan polysaccharide: from a century of past experiences to future prospects, Biotechnol. Adv. 34 (5) (2016) 827–844. [17] H.K. Sarilmiser, O. Ates, G. Ozdemir, K.Y. Arga, E.T. Oner, Effective stimulating factors for microbial levan production by Halomonas Smyrnensis AAD6 T, J. Biosci. Bioeng. 119 (4) (2015) 455–463. [18] H.K. Sarilmiser, E.T. Oner, Investigation of anti-cancer activity of linear and aldehyde-activated levan from Halomonas smyrnensis AAD6 T, Biochem. Eng. J. 92 (2014) 28–34. [19] E.A. Queiroz, Z.B. Fortes, M.A. da Cunha, H.K. Sarilmiser, A.M.B. Dekker, E.T. Öner, R.F. Dekker, N. Khaper, Levan promotes antiproliferative and pro-apoptotic effects in MCF-7 breast cancer cells mediated by oxidative stress, Int. J. Biol. Macromol.
635
Applied Surface Science 466 (2019) 628–636
N. Mihailescu et al.
[60] A.M. Sajjan, H. Premakshi, M.Y. Kariduraganavar, Synthesis and characterization of GTMAC grafted chitosan membranes for the dehydration of low water content isopropanol by pervaporation, J. Ind. Eng. Chem. (2014). [61] M.S. Bostan, Preparation and Characterization of Gene and Drug Delivery Systems Chemical Engineering Marmara University, İstanbul (2015) 145. [62] A. Poli, H. Kazak, B. Gürleyendağ, G. Tommonaro, G. Pieretti, E.T. Öner, B. Nicolaus, High level synthesis of levan by a novel Halomonas species growing on defined media, Carbohydr. Polym. 78 (4) (2009) 651–657. [63] A.F.D. Vasconcelos, R.F. Dekker, A.M. Barbosa, E.R. Carbonero, J.L. Silveira, B. Glauser, M.S. Pereira, M.d.L.C. da Silva, Sulfonation and anticoagulant activity of fungal exocellular β-(1→ 6)-D-glucan (lasiodiplodan), Carbohydr. Polym. 92 (2) (2013) 1908–1914. [64] I.N. Mihailescu, C. Ristoscu, A. Bigi, I. Mayer, Advanced Biomimetic Implants Based on Nanostructured Coatings Synthesized by Pulsed Laser Technologies, in: Antonio Miotello, Paolo M. Ossi (Eds.), Laser-Surface Interactions for New Materials Production Tailoring Structure and Properties Series: Springer Series in Materials Science, 130 2010, pp. 235–260. [65] Stefania Di Cio, Julien E. Gautrot, Cell sensing of physical properties at the nanoscale: mechanisms and control of cell adhesion and phenotype, Acta Biomater. 30 (2016) 26–48. [66] M. Vandrovcová, L. Bačáková, Adhesion, growth and differentiation of osteoblasts on surface-modified materials developed for bone implants, Physiol. Res. 60 (2011) 403–417. [67] H. Li, G. Zhang, J. Wu, C. Zhao, Y. Zhang, K. Shao, M. Han, H. Lin, J. Zhu, H. Na, A novel sulfonated poly (ether ether ketone) and cross-linked membranes for fuel cells, J. Power Sources 195 (19) (2010) 6443–6449. [68] L. Bacakova, E. Filova, F. Rypacek, V. Svorcik, V. Stary, Cell adhesion on artificial materials for tissue engineering, Physiol. Res. 53 (2004) S35–S45.
[51] V. Bansal, P.K. Sharma, N. Sharma, O.P. Pal, R. Malviya, Applications of chitosan and chitosan derivatives in drug delivery, Adv. Biol. Res. 5 (1) (2011) 28–37. [52] R. Riva, H. Ragelle, A. des Rieux, N. Duhem, C. Jérôme, V. Préat, Chitosan and chitosan derivatives in drug delivery and tissue engineering, Chitosan for Biomaterials II, Springer, 2011, pp. 19–44. [53] R.C. Goy, S.T. Morais, O.B. Assis, Evaluation of the antimicrobial activity of chitosan and its quaternized derivative on E. coli and S. aureus growth, Revista Brasileira de, Farmacognosia 26 (1) (2016) 122–127. [54] M. Ignatova, K. Starbova, N. Markova, N. Manolova, I. Rashkov, Electrospun nanofibre mats with antibacterial properties from quaternised chitosan and poly (vinyl alcohol), Carbohydr. Res. 341 (12) (2006) 2098–2107. [55] A.F. Martins, S.P. Facchi, H.D. Follmann, A.G. Pereira, A.F. Rubira, E.C. Muniz, Antimicrobial activity of chitosan derivatives containing N-quaternized moieties in its backbone: a review, Int. J. Mol. Sci. 15 (11) (2014) 20800–20832. [56] L. Cao, H. Zhang, C. Cao, J. Zhang, F. Li, Q. Huang, Quaternized chitosan-capped mesoporous silica nanoparticles as nanocarriers for controlled pesticide release, Nanomaterials 6 (7) (2016) 126. [57] F. Miao, Z. Guo, Y. Yi, S. Yang, The moisture absorption and retention abilities of quaternized carboxymethyl chitosan, Bioinformatics and Biomedical Engineering, 2009 ICBBE 2009. 3rd International Conference on, IEEE, 2009, pp. 1–3. [58] A. Visan, G.E. Stan, C. Ristoscu, G. Popescu-Pelin, M. Sopronyi, C. Besleaga, C. Luculescu, M.C. Chifiriuc, M.D. Hussien, O. Marsan, E. Kergourlay, D. Grossin, F. Brouillet, I.N. Mihailescu, Combinatorial MAPLE deposition of antimicrobial orthopedic maps fabricated from chitosan and biomimetic apatite powders, Int. J. Pharm. 511 (1) (2016) 505–515. [59] R. Deveikytė, R. Makuška, Quaternization of chitosan and partial destruction of the quaternized derivatives making them suitable for electrospinning, Chemija 24 (4) (2013) 325–334.
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