Introduction. Keywords Polyethylene Chitosan Electrospraying Antioxidant Stimuli-sensitive surface Food packaging

Iran Polym J DOI 10.1007/s13726-016-0421-0 ORIGINAL PAPER Polyethylene materials with multifunctional surface properties by electrospraying chitosan...
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Iran Polym J DOI 10.1007/s13726-016-0421-0

ORIGINAL PAPER

Polyethylene materials with multifunctional surface properties by electrospraying chitosan/vitamin E formulation destined to biomedical and food packaging applications Elena Stoleru1 · Silvestru B. Munteanu2 · Raluca P. Dumitriu1 · Adina Coroaba1 · Mioara Drobota˘1 · Lidija Fras Zemljic3 · Gina M. Pricope4 · Cornelia Vasile1 

Received: 27 April 2015 / Accepted: 2 February 2016 © Iran Polymer and Petrochemical Institute 2016

Abstract  A dual-bioactive layer based on antimicrobial chitosan and antioxidant vitamin E was immobilized onto PE surface using electrospraying as coating technique. Covalent bonding of the antibacterial/antioxidant layer was achieved through amide bonds or carbamate linkage using both 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysuccinimide or carbonyldiimidazole coupling agents, respectively. The chitosan/vitamin E formulation was characterized by rheological measurements. The vitamin E addition in chitosan matrix leads to changes in chitosan rheological properties, such as viscosity decrease with increasing vitamin E content, change of the gel-like behavior to a fluid-like behavior, which further influences the electrospraying process and deposited coating morphology. The new stratified hybrid materials with improved properties have been characterized by different techniques as attenuated total reflectance-Fourier transform IR spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS), polyelectrolyte and potentiometric titration, Electronic supplementary material  The online version of this article (doi:10.1007/s13726-016-0421-0) contains supplementary material, which is available to authorized users. * Cornelia Vasile [email protected] 1

“Petru Poni” Institute of Macromolecular Chemistry, Romanian Academy, 41A Gr. Ghica Voda Alley, 700487 Iasi, Romania

2

Faculty of Physics, “Al. I. Cuza” University, Bvd. Carol I, 11, Iasi, Romania

3

Faculty of Chemical Engineering, University of Maribor, Smetanova ulica 17, 2000 Maribor, Slovenia

4

Veterinary and Food Safety Laboratory, Food Safety Department, Iasi, Romania





contact angle titration, scanning electron microscopy (SEM) and antibacterial and antioxidative tests. The electrosprayed bioactive coatings exhibit antibacterial, antioxidant and pH responsive activity. The pH responsiveness was evidenced by switching from hydrophilic to hydrophobic surface at pH ≈ 6. The chitosan/vitamin E modified PE substrate inhibited the growth for three different bacterial strains (Gram-negative and Gram-positive) and presented good antioxidative properties, acting as DPPH radical scavenging surfaces. Moreover, the new obtained materials present good stability and maintain their antioxidative capacity even after subjecting to desorption in harsh medium because of relative strong electrostatic and hydrogen bonds interactions between components of the formulation. The obtained materials can find application in food packaging or in medical field where synergistic action of these bioactive compounds is required. Keywords  Polyethylene · Chitosan · Electrospraying · Antioxidant · Stimuli-sensitive surface · Food packaging

Introduction Active packaging is a class of packaging which interacts directly with the packed material and/or its surroundings to improve one or more aspects of the quality or safety properties of the products [1, 2]. A number of techniques have been explored to combine active compounds into packaging systems and then to impart them antioxidant and antibacterial properties. Antimicrobial coatings gathered much attention lately due to the boost in implant technology, and their clinical usage, on one hand, and the medical device related infections, on the other hand [3]. Moreover, coatings combining antimicrobial effects together with

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antioxidant activity were successfully tested for food protection studies both in vitro and in vivo [4]. Polymers are attractive materials for a large range of applications including biocompatible materials, coatings, thin-film technology, microelectronic devices and others. Polyethylene (PE) is produced in several grades as low density (LDPE), linear low density (LLDPE) and high density (HDPE) with different properties (amorphous-crystalline fraction, mechanical properties, transparency, heat resistance, chemically inertness, moisture barrier) and applications such as packaging, construction of particular portions of implants used for hip and knee replacements and also other uses as biomaterials [5]. In most of biomedical applications and food packaging high biocompatibility and good interaction with environment is required. These characteristics are achieved (among other procedures) by surface modification through chemically bonding of biocompatible macromolecules, being known that PE is an inert polymer. In this case, surface preactivation followed by a multi-step bonding procedure is necessary [6]. Surface functionalization provides desired changes in physical properties of the substrate surface (e.g., wettability, adhesion, and biocompatibility) [7]. Although noncovalent adsorption may be useful in some applications, covalent immobilization provides the most stable bond between the compound and the functionalized polymer surface [8]. Among the functionalization techniques applied to polymer surface modification, plasma treatment offers some advantages as it alters only the surface layers of the substrate, but its bulk properties remain unaffected [9]; it is a solvent free method and the byproducts being environmental friendly, the surface modifications can be tailored according to the application by changing plasma parameters [10]. The paper presents the techniques for obtaining and characterization of new stable multifunctional bioactive (antimicrobial, antioxidant, biocompatible) and pH responsive coatings based on chitosan and vitamin E on polyethylene substrate. The subject of the study is in line with the modern research in the field. Although chitosan/vitamin E formulations were used in the past [11] in this work new functional groups are implanted on the polyethylene surface, after corona pretreatment, for chitosan/vitamin E anchoring. The electrospray/electrospinning technique was used for bioactive layer deposition of the mixture of chitosan/vitamin E. The advantages of the electrospraying technique are high material deposition efficiency, simplicity of experimental set-up, great flexibility in the choice of starting materials, a broad range of very thin surface layers with various compositions and morphologies that can be obtained with minimum material consumption [12, 13]. Deposition of a very thin coating with complex composition, such as chitosan/vitamin E combination, leads to the obtaining of a multifunctional surface. The obtained material combines

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the antibacterial, antifungic, bioadhesivity, biocompatibility, biodegradability of chitosan [14, 15] with the biological functions and antioxidative activity of vitamin E (it prevents cell membrane damage by inhibiting peroxidation of membrane phospholipids and disrupting free radical chain reactions induced by formation of lipid peroxides) [16] which could be very useful in most of polyethylene applications. Different methods have been applied to produce hybrid materials based on chitosan and synthetic polymers such as grafting [17], chemical functionalization [18], wet spinning [19], chemical cross-linking [20], casting [21], melt blending [22] and electrospinning [23]. In this paper chitosan and vitamin E have been immobilized onto corona pretreated PE using the most efficient coupling agents (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/ NHS) and carbonyldiimidazole (CDI)) and coating method (as electrospraying/electrospinning). This two-step procedure was not applied until now. The chitosan/vitamin E formulation was characterized by rheological measurements. The physical–chemical characterization of the deposited thin coatings was done by different techniques as attenuated total reflectance-Fourier transform spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS), polyelectrolyte titration, potentiometric titration and scanning electron microscopy (SEM). Furthermore, the pH responsiveness (by contact angle titration), antibacterial and antioxidative properties were tested. The new developed stratified composites materials based on polyethylene, chitosan and vitamin E, possessing pH responsiveness, antibacterial and antioxidative properties are recommended for different applications such as active food packaging, biomedical devices and hospital equipments.

Experimental Materials Polyethylene, with a 0.02 mm thickness, purchased from SC LORACOM SRL, Roman, Romania. Medium molecular weight chitosan (CHT) with 200–800 cP viscosity in 1 % acetic acid and 75–85 % deacetylation degree was purchased from Sigma-Aldrich, USA. Vitamin E (tocopherols) is a fat-soluble vitamin, which functions solely as a membrane-bound antioxidant that occurs in food as tocopherols or tocotrienols, α-tocopherol being the most biologically active form of vitamin E. Vitamin E (VE)—synthetic α-tocopherol ≥96 % (HPLC) purity (Sigma-Aldrich, USA) and glacial acetic acid (99.5 %) from Chemical Co., Iasi, Romania were used. Water-soluble carbodiimide cross-linkers for zero-length, carboxyl-to-amine conjugation EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide

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containing vitamin E in the range of 0.5–3 wt%. The solution of 75 mM EDC + 15 mM NHS in water was used to activate the carboxylic groups formed at the PE surface after corona pre-treatment. The CDI solution (20 mM concentration) prepared in ethanol with a water content smaller than 0.1 %, could activate both carboxyl and hydroxyl groups, possible formed at the PE surface after corona pre-treatment. Oscillatory and rotational rheological measurements

Scheme 1  Schematical representation of the electrospraying set-up

hydrochloride) and NHS (N-hydroxysuccinimide), both with purity >98 %, from Sigma-Aldrich (USA) were also utilized. N,N′–Carbonyldiimidazole (CDI) reagent (SigmaAldrich, USA) is a highly active carbonylating agent that contains two acylimidazole leaving groups. CDI was used to activate carboxylic or hydroxyl groups for conjugation with other nucleophiles, creating either zero-length amide bonds or one-carbon-length N-alkyl carbamate linkages between cross-linked molecules [24]. Electrospraying technique The electrospraying system used was a conventional one, consisting of a direct current high voltage supply, a rotating metal plate collector, and a syringe oriented with the low diameter needle perpendicular to the metal plate (Scheme  1). The high direct voltage (0–30 kV) is applied between the metal plate and syringe needle. The distance between the tip of the needle and the metallic plate can be set in the 4–40 cm range. A more detailed description of the apparatus was presented in Munteanu et al. [25]. Functionalization of PE surface After corona activation (frequency 30 kHz, interelectrodes distance 7 mm, discharge power 45 kJ/m2) and air exposure both oxygen containing functional groups (carboxyl, carbonyl, hydroperoxide, peroxide, etc.) and radicals are implemented on PE surface decreasing hydrophobicity and improving the adhesion with bioactive mixture and also act as active sites for further modification. Chitosan/ vitamin E formulation was electrosprayed onto untreated and corona treated PE films (Scheme 1) and covalently bound (immobilized) onto corona-activated surface using coupling agents (EDC and NHS or CDI). Chitosan solution used for electrospraying had a 2.3 wt% concentration, prepared in twice-distilled water with 70 % acetic acid,

A Physica MCR 301 rheometer (Anton Paar, Austria) equipped with cone-plate measuring system of 50 mm diameter was used. The measurements on chitosan and chitosan/vitamin E solutions with different vitamin E concentrations (0.5–3 wt%) were performed at constant temperature (25 ± 0.1 °C) at a 0.5 mm gap. Strain sweep tests were performed previously, at 25 °C at 10 rad/s over the strain range 0.01–100 % to estimate the linear viscoelastic range (LVE); the results showed a LVE regime up to a strain value of 0.01 %. Oscillatory shear tests were made in the frequency range 0.1–100 s−1. Steady state shear flow tests were also carried out on the same solutions, the shear rate ranged 0.01–100 s−1. To prevent dehydration, a thin layer of low-viscosity silicone oil has laid on the air/sample interface. All presented data are the average of at least three determinations. ATR‑FTIR The ATR-FTIR spectra were recorded by means of a spectrometer Bruker VERTEX 70 (Germany) in absorbance mode. Background and the sample spectra were obtained in the 600–4000 cm−1 wavenumber range and collected as 64 co-added scans at 2 cm−1 resolution. Five spectra were recorded for each sample. In the case of overlapping bands, deconvolution was performed using OPUS program. Spectra of samples were deconvoluted with a smoothing filter of 25 %. Each spectrum was baseline corrected and the absorbance was normalized between 0 and 1. XPS XPS measurements were performed on a KRATOS Axis Nova (Kratos Analytical, Manchester, UK) using AlKα radiation, with 20 mA current and 15 kV voltage (300 W), and base pressure of 10−8–10−9 Torr in the sample chamber. The incident monochromated X-ray beam was focused on a 0.7 × 0.3 mm area of the surface. The XPS survey spectra for the samples were collected in the range of −10 to 1200 eV with a resolution of 1 eV and a pass energy of 160 eV. The high resolution spectra for all the elements identified from the survey spectra were collected

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using a pass energy of 20 eV and a step size of 0.1 eV. The Casa XPS software was used for background subtraction (Shirley-type), peak integration, fitting and quantitative chemical analysis. All binding energies were referenced to the C1s peak at 285 eV. The resolution for the measurements of the binding energy was about 0.2 eV. Potentiometric titration A two-burette instrument (Mettler Toledo) equipped with a combined glass electrode (Mettler T DG 117) was used. The burettes were filled with 0.1 M HCl (Fluka, analytical) and 0.1 M KOH (Baker, Dilut-it). All the solutions were prepared with deionised water with very low carbonate content (≤10−6) which was achieved trough boiling and subsequent cooling under nitrogen atmosphere. The PE films coated with chitosan/vitamin E and the reference PE were titrated in forward and back runs between pH 2.8 and 11. The titration experiments were carried out at 0.1 M ionic strength, set to its appropriate value with KCl (Riedel-de-Häen, Germany). Under same conditions the blank HCl–KOH titration was performed. Polyelectrolyte titration Polyelectrolyte titrations were carried out in an aqueous media at two different pH values (pH 3.6 and 6.5), adjusted by 0.1 M HCl. For a typical measurement a 50 mg of chitosan/vitamin E-electrosprayed PE sample were immersed for 48 h in 50 mL of aqueous solution adjusted at the two pHs 3.6 and 6.5. The solution was filtrated at pre-established time and filtrates were further used for polyelectrolyte titration. One mL of filtrated solution pipetted into a titration vessel and 1 mL of 0.1 mM indicator Toluidine Blue was added. The solution diluted with distilled water to a volume of 40 mL. A Mettler Toledo DL 53 titrator (Switzerland) with a 10 mL burette was used for incremental addition of the polyelectrolyte titrant (polyethylenesulphonate sodium salt —PES-Na; C = 10 mM). The absorbance measured a s a potential change in mV, using a Mettler Toledo Phototrode DP660 (Switzerland) at a wavelength of 660 nm. The concentration of protonated amino groups determined from the equivalent volume of PES-Na solution added, detected as the steepest rise of the absorbance versus volume of PVS, and by assuming a 1:1 binding stoichiometry of vinylsulfonate to ammonium groups. The experiments have performed at least three times at room temperature and average of the measurements has used in results discussion.

non-animal origin namely Salmonella spp., Listeria spp. and Escherichia coli. These tests are also valid for medical applications of materials. The lyophilized ATCC cultures: Salmonella typhymurium - 14028, Listeria monocytogenes - 7644 and Escherichia coli - 25922 which were purchased from American Type Culture Collection (Rockville, MD) were used as microorganism strains in this study. The ATCC cultures were reconstituted according to the requirements of specific standards such as: SR EN ISO 11133/2014—Microbiology of food, animal feed and water—Preparation, production, storage and performance testing of culture, ILAC G9/2005—Guidelines on the selection and use of reference materials and SR EN ISO 7218-A1/2014—Microbiology of food and animal feeding stuffs—General requirements and guidance for microbiological examinations—Amendment 1. The lyophilized culture has subcultured to obtain replicate reference stock cultures and further the reference stock culture subcultured to obtain the working stock culture. From this working stock culture was obtained bacterial suspensions of 0.5 McF (measured with a densitometer). The obtained suspensions were serial diluted to achieve concentrations of about 102–103 UFC/0.1 mL that have used for testing the prepared polymer samples. Sterilization of the chitosan/vitamin E modified-PE samples has made in autoclave at 110 °C and 0.5 bar for 20 min. The samples were placed in Petri dishes and incubated at 37 °C. After 24 and 48 h, using sterile tampons moistened in peptone physiological serum, were collected the specimens from the test surfaces and were seeded on the surface of specific culture media: XLD—xylose lysine desoxycholate agar for Salmonella typhymurium; ALOA— agar listeria ottaviani and agosti for Listeria monocytogenes; and VRBG—violet red bile glucose agar in the case of E. coli. The Petri dishes with inoculated culture media were incubated at 37 °C and after 24 h the specific colonies of each bacterial species were counted. All experiments were performed comparatively with alimentary commercial PE foils. The samples notation (code) includes the abbreviation of the components and method of obtaining, for example: PEcor, EDC+NHS, CHT/VE or PEcor, CDI, CHT/VE is corona treated PE film, immersed in solution of coupling agents 1-ethyl-3-(-dimethylaminopropyl) carbodiimide (EDC) + N-hydroxysuccinimide (NHS) or carbonyldiimiazole (CDI) and further coated with chitosan/vitamin E mixture by electrospraying. DPPH radical scavenging assay

Antibacterial tests Antibacterial tests have been performed using specific bacteria usually tested for food products both of animal and

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The radical scavenging activity (RSA) of chitosan/vitamin E coated PE films has been measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which is considered to

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be a good in vitro model widely used to assess free radical scavenging efficacy in a relatively short time. The antioxidant and radical scavenging activity of α-tocopherol (vitamin E) is well known and studied [26, 27]. The DPPH radical is more stable than the in situ-generated superoxide anion and hydroxyl radical. Comparing EC50 (50 % radical scavenging ability) values of 54.16 and 89 μg/mL for scavenging activities of α-tocopherol for hydroxyl and DPPH radicals, respectively, showed that DPPH radical is much suitable to test antioxidant activity of surface layer deposited on PE surface. Approximately 100 mg of CHT/VE coated film was placed in a flask with 10 mL methanol containing 10−4 M DPPH. The reaction mixture was left to stand for 30 min at room temperature in the dark. The scavenging activity of samples was estimated by measuring the absorption of the mixture at 515 nm, using a UV–Vis spectrometer (Cary 60-UV–Vis Spectrophotometer, USA) which reflected the amount of DPPH radicals remaining in the solution. The control sample was the same DPPH solution in the absence of the coated-film. The RSA of the chitosan/vitamin E coated films was calculated according to the following equation:   Asample × 100 RSA (%) = 1 − (1) Acontrol where Asample represents the absorbance of the sample solution and Acontrol represents the absorbance of DPPH solution without the addition of the film. Contact angle titration Images of buffered liquid droplets on the substrates were taken using a contact angle goniometer CAM 200, KSV Finland. Contact angle titrations were performed by measuring no less than five sets of contact angles at each pH value for 1 μL drops.

Results and discussion Influence of rheological properties of chitosan/vitamin E formulation on electrospraying ability Small amplitude oscillatory shear measurements were performed in the short LVE range (0–100 rad/s). In addition, rotational tests were used to determine the viscosity of the solutions as a function of shear rate. The shape of the viscosity curves clearly shows decreasing viscosity values with increasing frequency and respectively, shear rate, indicating that all solutions behave as non-Newtonian (pseudoplastic), shear-thinning fluids. In Fig. 1a, the steady shear and complex viscosities for the chitosan and chitosan/VE solutions at various

Fig. 1  Comparison of the complex viscosity (η*) and steady shear viscosity (η) data of chitosan solutions with various vitamin E contents (a) and effect of vitamin E content in chitosan solutions on storage (G′) and loss (G″) moduli as functions of frequency at 25 °C (b)

concentrations are compared. These viscosities showed some differences mainly for neat chitosan solution, visible at low frequency and shear rate, which become negligible at higher deformation rate. The most significant decrease in viscosity with shear rate was recorded for chitosan solution. In case of neat chitosan solution, the viscosity varied from 4250 to 2.49 Pa.s, while for the chitosan with 3 wt% VE the viscosity varied from 22.7 to 0.64 Pa.s. The oscillatory tests were performed mainly to determine the dynamic moduli, i.e., storage (G′) and loss moduli (G″) in the linear viscoelastic region. Figure 1b illustrates the dynamic moduli G′ and G” as functions of frequency, in terms of vitamin E content/concentration in the chitosan/ vitamin E solutions. Both G′ and G″ moduli and viscosity decreased with increasing vitamin E concentration in solution, the highest values for storage (G′) and loss moduli (G″) being determined for the neat chitosan solution. The viscosity values measured at 2.5 rad/s decreased from

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22.2 Pa s for chitosan solution to 2.88 Pa s for the solution with 3 wt% vitamin E in composition. At lower angular frequencies, the G′ value corresponding to neat chitosan was higher than G″, indicating a gel-like behavior of the solution, while for the samples containing vitamin E the values of the loss modulus were generally higher than those of the storage modulus, corresponding to a fluid-like behavior. With increasing frequency above 10 rad/s the curves were overlapped and no significant differences between samples were noticed. The reduction of the elasticity may result from the decrease in the number of interactions and entanglements between the chitosan molecules, due to the increased chain flexibility and disentanglement at high shear rates. The pronounced shear-thinning effect can be related to the structure of the polymer chains in solution. Without an external load, each single macromolecule can be found in the shape of a three-dimensional coil because this is the lowest energy state. During the shear process, however, the molecules are more or less oriented parallel to the direction of shear, resulting in elongation, which lowers their flow resistance and results in a decrease of the bulk viscosity [28]. The zero-shear viscosity was determined from the shear data using the Carreau-Yasuda model. The zero-shear viscosity (η0) decreased significantly with increasing vitamin E concentration, from 4850 Pa.s for neat chitosan solution to 35 Pa.s for CHT/3 %VE. Chitosan/vitamin E solution rheological behavior is dependent on the solvent used. In acetic acid, a decrease in viscosity was found in this study, while for solutions of CHT/VE in lactic acid the viscosity increases with increasing VE concentration [28]. Rheological behavior of chitosan/vitamin E solutions influence the morphology of deposited coating (Supporting Information). The addition of small quantities of vitamin E to solutions of chitosan transforms the process from electrospraying to electrospinning leading to the formation of thin coating films with diverse nanostructures, from nanospheres to three-dimensional coatings via connectiveparticle formation or nanofibres with increasing vitamin E quantity and consequently decreasing mixture solution viscosity (Supporting Information). A zero-shear rate viscosity of 35 Pa.s seems to be the most suitable for nanofibres formation. Structural and physical–chemical characterization of chitosan/vitamin E electrospraying coating In ATR-FTIR spectra of untreated and corona treated PE films coated by electrospraying of CHT/VE, all bands of the components are presented (Fig. 2a). The IR absorption bands of α-tocopherol (Fig. 2a—Spectrum 6) are at 3463 cm−1 assigned to OH, at 2925 and 2867 cm−1 for asymmetric and symmetric stretching vibrations of the CH2

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Fig. 2  ATR-FTIR spectra of chitosan/vitamin E-coated PE surfaces by electrospraying: a (1) PE; (2) PE,CHT/VE; (3) PEcor,CHT/VE; (4) PEcor, EDC+NHS, CHT/VE; (5) PEcor, CDI, CHT/VE; (6) VE; (7) CHT; b infrared spectra in the wavenumbers range 1800– 1700 cm−1 of chitosan matrix with different vitamin E contents (0.5 and 3 wt%)

and CH3 groups, at 1459 cm−1 for phenyl skeletal bending, at 1377 cm−1 for methyl symmetric bending, at 1261 cm−1 for −CH2, at 1085 cm−1 for plane bending of phenyl and at 918 cm−1 for trans =CH2 stretching vibration [29]. The FTIR spectrum of chitosan (Fig. 2a—Spectrum 7) shows a broad −OH stretching absorption band between 3660 and 2983 cm−1 and the aliphatic C–H stretching between 2983 and 2790 cm−1 [30]. The band corresponding to νOH vibration has a maximum at 3352 cm−1, because this band is quite broad has covered the characteristic N–H stretch band for chitosan. Another major absorption band with a maximum at 1591 cm−1 belongs to free primary amino group bending (νNH2) at C2 position of glucosamine, which is a major group present in chitosan. Peak at 1650 cm−1 is assigned to acetylated amino group in chitosan, which indicates that the sample is not fully deacetylated according to Sigma-Aldrich specifications. The band

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occurring at 1376 cm−1 is ascribed to the −C–O stretching of primary alcohol groups (−CH2-OH). The absorption bands at 1151 cm−1 (anti-symmetric stretching of the C–O–C bridge), 1064 and 1028 cm−1 (skeletal vibration involving the C–O stretching overlapped with C–N stretching vibration) are characteristics of its saccharide structure [31]. In the IR spectrum of the CHT/VE-coated corona untreated PE, very weak bands characteristic of chitosan can be identified (Fig. 2a—Spectrum 2). In this case, the electrosprayed chitosan is just physically adhering to the surface, while after CHT/VE deposition onto corona activated PE, the IR bands of chitosan become well defined and with higher intensities, therefore the CHT/VE coating is significant and presents stability only after corona pretreatment of the surface. In the CHT/VE-coated PE spectrum, the characteristic amine peak and amide I peak of the acetyl group of chitosan appear at 1597 and 1651 cm−1, respectively. These bands have shifted to lower wavenumbers in the spectra for CHT/VE-covalently bonded PE (Fig.  2a—Spectra 4 and 5). These results indicate strong interactions between chitosan/vitamin E formulation after coupling reaction onto corona treated PE. When using EDC+NHS and CDI coupling agents, distinguishable changes in band patterns are observed. In this case new amide bonds formed lead to new absorption bands in the IR spectra at 1637 cm−1, assigned to stretching vibration of –C=O (amide I), and at 1568 cm−1 which corresponds to the δNH (in-plane deformation vibrations), when using EDC and NHS coupling agents [32]. In the case of CDI coupling route, the amide I bands are located in the IR spectrum at 1645 and 1564 cm−1. These spectral differences assess the chemical bonding of chitosan/ vitamin E mixture onto PE surface by two steps-procedure (corona pre-activation followed by coupling reaction with both agents). The IR absorption bands of vitamin E overlap with those characteristic to chitosan and PE. To evidence the presence of vitamin E in the composite samples and possible interactions between formulation components or with substrate, the deconvolution of FTIR spectra was performed for three spectral regions: 2600–3600, 1500–1750 and 1200–1500 cm−1 (Supporting Information—Fig. S2a–f). Initially PE spectrum was subtracted from the spectra of the chitosan and vitamin E deposited onto PE substrate and further deconvolution was performed. By deconvolution of the absorption band in the 3600– 2600 cm−1 spectrum region numerous bands were found. The bands from 3518 cm−1 for chitosan and 3529 cm−1 for CHT+VE mixture are attributed to free OH groups. The peak at 3440 cm−1, which can be assigned to internally bonded OH stretching vibration, shifts to 3469 cm−1 by the vitamin E addition. Furthermore, after VE addition a new

band appears at 3416 cm−1 which can be related to interand intra-molecular bonding of hydroxyl groups [33, 34] and stretching vibration of secondary amine groups (N–H) present in chitosan. Moreover, another new band found in the spectrum at 3023 cm−1 can be assigned to C–H stretching vibration of the double bond (=CH) [35] which is characteristic to vitamin E. By deconvolution of the 1500 and 1200 cm−1 IR band region, numerous bands were found especially when vitamin E is added attesting vitamin E presence such as the band at 1441 cm−1 attributed to phenyl skeletal bending and 1226 cm−1 which can be assigned to the bending vibration of the −OH phenolic group [36]. Furthermore, when analyzing the deconvolution of the 1750–1500 cm−1 IR region considered as “fingerprint region”, six bands are observed for electrosprayed chitosan, when adding vitamin E into formulation, two new bands at 1724 and 1546 cm−1 are appeared. The 1724 cm−1 band can be assigned to the carbonyl stretch of vitamin E, which possible results from −OH group conjugation with phenyl ring, knowing that phenol compounds exhibits keto-enol tautomerism [38]. The band at 1562 cm−1, for primary amine bending, is shifted to 1546 cm−1 most probably due to the electrostatic interactions occurring between partially ionized −NH2 groups of chitosan with phenolic OH groups of vitamin E in acidic medium [5]. The interaction between chitosan and vitamin E is mostly electrostatic and trough hydrogen bonds, as suggested also by other authors [37, 39]. Based on these data the phenomena occurring during surface treatment of PE with the two bioactive agents can be represented as in Scheme 2. By interaction between chitosan and vitamin E a relative stable association was formed by electrostatic forces and hydrogen bonding— Scheme 2a. When pretreated PE film is coated by electrospraying with the CHT/VE mixture, the functional groups implemented by corona discharge may react with chitosan amino groups, forming stable amide bonds [40] or N-alkyl carbamate linkage depending on the coupling agent used (Scheme 2b). When comparing the IR spectra of PE coated with mixture of chitosan/vitamin E, which contains different amounts of vitamin E, a shoulder at 1740 cm−1 is distinctive for vitamin E, noticeable even at a low content (0.5 wt%). A slightly increase in intensity of this shoulder is observed at higher content of VE (3 wt%) (Fig. 2b). In the followings, only results on the chitosan/0.5 wt% VE formulation are presented. From the XPS measurement, C1s core level spectra of the PE and CHT/VE-coated/covalently bonded samples (Fig. S5 in Supporting Information) the variations of the corresponding areas (Table 1) were considered to evidence differences between samples. The O/C and N/C ratios of

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Scheme 2  Possible reaction route between chitosan and vitamin E in acidic medium (a) and corona-activated PE surface using EDC+NHS and CDI coupling agents (b)

Table 1  C1s components content and assignments of corona treated PE films and coated with chitosan/vitamin E Sample code

Atomic ratios

Contribution of C1 s components (%)

Atomic ratio

O/C

N/C C1 (C–C, C–H) C2 (C–NH2 and/or C–O–C) C3 (C–O)

PE PEcorona PE,CHT/VE PEcor,CHT/VE PEcor,EDC+NHS,CHT/VE PEcor,CDI,CHT/VE PEcor,EDC+NHS,CHT/ VE, pH 3.5

0.01 0.10 0.29 0.34 0.34 0.37 0.36

0.01 0.06 0.06 0.07 0.07 0.08

PEcor,CDI,CHT/VE, pH 3.5

0.40 0.10 26.2

100.0 93.3 42.5 48.3 36.2 44.3 48.0

C4 (N–C=O) C5 (O=C–O)

C4/C2

– 2.8 (C–O) 23.8 15.7 19.1 17.6 13.8

– 3.9 (C=O) 21.1 24.4 27.3 23.1 26.7

– – 8.5 7.8 11.6 9.7 9.2

– – 4.1 3.8 5.8 5.3 2.3

– – 0.35 0.49 0.61 0.55 0.66

43.9

20.3

6.9

2.6

0.16

The underlined elements represent core-ionized atom

the modified samples varied obviously with the sequential process of corona treatment and coating/covalently binding of CHT/VE mixture from 0.011 to 0.4 and from 0.01 to 0.1, respectively. To evaluate the stability of the deposited coating, XPS spectra recorded also for the covalently bonded samples after subjecting them to the action of an acidic solution with pH 3.5. The elemental composition data (Supporting information—Table S1) proved that the CHT/ VE coating is stable even in harsh environment because of its irreversible binding onto corona treated PE substrate. The C1s high-resolution spectrum of PE presents two peaks at 285.0 and 285.6 eV (Supporting information— Fig. S5a), the first one assigned to C–C and/or C–H, being the most intense, and the second peak attributed to C–O, possibly due to a slight surface oxidation. The C1s spectra of the other obtained samples can be curve-fitted with

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three or five peak components from chemically non-equivalent carbon atoms mainly bonded to oxygen, in the case of corona discharge activated sample, and to nitrogen for samples coated with CHT/VE. The C1s high-resolution spectrum of corona treated PE film can be curve-fitted with three peak components (Supporting information—Fig. S5b). One major peak at 285.0 eV (C1) is assigned for C–C/C–H, and two other at 286.2 eV (C2) and 287.3 eV (C3) are corresponding to C–O and C=O bonds, respectively. Each of the chitosan/vitamin E-coated C1s core level spectra were deconvoluted into five peaks (Supporting information— Fig. S5c–f), namely: 285.0 eV (C–C and C–H), 286 eV (C-NH2 and/or C–O–C), 286.8 eV (C–O), 288 eV (C with O, C=O group and amide bond) (C4), and 289 eV (ester group) (C5), while for chitosan-coated PE the C1s

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core level spectrum can be deconvoluted only in three peaks [40]. The other two peaks reveal the vitamin E contribution. The content change of each chemical component— (Table  1)—consists in significantly decrease of the C–C bond percentage after corona treatment, and at the same time, most of the oxygen containing polar groups C–O, C=O content increase at the surface of the corona treated polymer surfaces. These results indicate that some of the C–C/C–H bonds at the polymer surface cleave by the action of corona treatment, and the resulted radical active species recombines with oxygen and possible some nitrogen atoms to form new stable bonds. One of the ways to achieve CHT/VE covalently bound onto corona treated PE surface is by amide bond formation between surfaces containing oxygen functionalities and chitosan amino group by means of using various coupling agents (e.g., EDC+NHS or CDI). The XPS atomic ratios C4/C2 (N–C=O/C-NH2) gives information about amide bond formation. The C4/C2 atomic ratio of all chitosan/vitamin E coated sample increases after using both coupling routes (Table 1) showing a higher increase for EDC+NHS case. This means that the grafting degree of chitosan/vitamin E onto corona functionalized PE is higher when using EDC+NHS system than using CDI which is directly correlated with the surface layer stability. Furthermore, an increase in the atomic percentage of C5, assigned for the C atom involved in ester bond, is noticed for PEcor,EDC+NHS,CHT/VE. This may be due to the interaction between the carboxylic group formed onto PE surface after corona treatment and air exposure, and hydroxyl groups of chitosan. The above-mentioned data emphasize again that EDC+NHS coupling system is more effective than CDI. The presence of nitrogen in XPS spectra of the samples prior subjected to desorption was identified only in the case of the samples obtained by covalent coupling (Supporting information—Table S1). In this case, the stability of CHT/VE coating on top of PE substrate is proved. Therefore, the functionalization of the substrate influences even the efficiency of the electrospraying deposition. No charge was detected in the reference material (PE) and vitamin E—due to its high pK value–hence these components do not contribute to the protonation/deprotonation process. The presence of chitosan brings into the investigated samples amino groups that can be protonated. The total charge amount of each investigated sample was calculated from the plateau level of charging isotherms (Fig. 3a). The PEcor,EDC+NHS,CHT/VE sample presents the highest content of charged amino groups, as it was concluded also from XPS data shown above.

Fig. 3  Charging isotherms (a) and desorption kinetic curves at pH 3.6 (b) of chitosan/vitamin E-coated PE films Table 2  Percentage of amino group desorbed from chitosan/vitamin E polymer surfaces after 48 h Sample code

PE/CHT+VE PEcor/CHT+VE PEcor/CDI/CHT+VE PEcor/EDC+NHS/CHT+VE

Desorbed amino groups (%) pH 3.6

pH 6.5

100 19.64 17.6

96.5 62 62.33

20.4

32.02

Chitosan/vitamin E desorption from the PE surface was evaluated using polyelectrolyte titration and the amount of amino groups associated with desorbed chitosan/vitamin E (Fig. 3; Table 2). In Fig. 3b, the desorption curves at pH 3.6 of chitosan/vitamin E coated PE films are presented. The main characteristic of desorption behavior for all samples is that when pH was 6.5 (Supporting information—Fig. S4) the CHT/VE mixture desorption was slower than the case when the pH of the desorption bath was 3.6. This behavior is because at acid pH (3.6) all the accessibly primary amino groups of chitosan are

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protonated, i.e., NH3+ and passed easier in solution. At acidic pH the percentage of the desorbed chitosan/vitamin E is higher. The desorbed amount of CHT/VE is dependent on the initial amount deposited onto surface. In the case of PEcor/EDC+NHS/CHT+VE sample the highest amount of chitosan/vitamin E was bonded (78 mmol/kg) and the lowest was desorbed (32 %). Using EDC+NHS coupling system an irreversible binding of chitosan/vitamin E mixture onto PE sample is achieved. The spectra recorded for the samples after desorption qualitatively reveal the presence of chitosan/vitamin E onto polymer surface, even after 48 h period of desorption, only when PE was corona pretreated and especially when coupling reaction was applied for bioactive mixture immobilization (Supporting information—Fig. S3). Antibacterial activity of the chitosan/vitamin E‑coated PE films The chitosan/vitamin E-coated PE films were tested against two gram-negative bacteria, namely Salmonella Typhimurium and Escherichia coli, and one gram-positive bacterium, Listeria monocytogenes and the results are presented in Fig. 4a. Electrospraying of chitosan/vitamin E formulation imparts antibacterial activity to the polymeric substrate, PE. It is known that the protonated amino groups from chitosan structure play a crucial role in the antibacterial activity. Previous studies showed that only corona treatment is not enough to obtain a stable chitosan coating therefore the chemical binding of this one onto the surface is mandatory. When using EDC+NHS and CDI coupling agents, stable chitosan/vitamin E layer onto the PE surface is obtained and microbiological activity of chitosan is preserved. The EDC+NHS coupling system is more efficient for chitosan covalent binding than CDI. Even though the first coupling system is more covalent binding-effective, the second one mediates the binding of fewer primary amino groups from chitosan, groups that are involved in the antibacterial activity. The data presented in Fig. 4a reveal that the antibacterial activity of the covalently bounded chitosan/vitamin E layer decreases when compared with the physical adsorbed one, but still maintains its antibacterial property against some bacteria. By electrospraying deposition of chitosan/vitamin E formulation onto PE surface, a coating of approximately 8.5 nm (which was determined through cross section examination by fracture analysis using scanning electron microscopy—data not shown) is formed. The sample PEcor,CHT/VE presents antimicrobial activity against all three tested bacteria and it can be concluded that it is necessary only a very thin chitosan layer (in the micrometer range) to impart antibacterial activity to PE.

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Fig.  4  a The antibacterial activity of chitosan/vitamin E-coated PE surfaces and b DPPH radical scavenging activity (RSA) of the CHT/ VE-coated PE films for increasing concentrations of α-tocopherol

Antioxidative activity of chitosan/vitamin E‑coated PE films In Fig. 4b, the DPPH radical scavenging activities of CHT/ VE-coated PE substrates with different contents of vitamin E are presented. For RSA tests was used a DPPH concentration of 10−4 M and 100 mg of sample. In this experimental conditions the RSA increases with increasing α-tochopherol content, as it is logical, knowing that the reaction is equimolar. In this case, even the coating having incorporated 0.5 % vitamin E would achieve a 100 % RSA by increasing the amount of sample. DPPH radical scavenging activity was determined also for covalently bound chitosan/vitamin E samples after subjecting them to desorption in acidic medium. A radical scavenging activity (RSA) of 18.5 % (after 30 min) was obtained denoting that vitamin E remains incorporated into chitosan-based formulation, demonstrating the achievement of a stable dual-bioactive coating on top of the PE substrate.

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pH responsiveness evaluation pH responsiveness of chitosan/vitamin E modified polyethylene surface was evidenced by contact angle measurements using buffered solutions with pH varying in the 2–11 range. The contact angle of the polyethylene surface remains constant over the entire pH range (Fig. 5a). The titration curves for chitosan/vitamin E coated PE are sigmoid. Until pH 6 the substrate surface coated with chitosan/vitamin E is hydrophilic, after which the contact angle sharply increases and passes to a hydrophobic region (θ >90°) indicating a more pronounced pH sensitivity. When compared with the system containing only chitosan it is noticed that the critical pH shifts slightly to higher pH values by adding vitamin E in the system (Fig. 5b) suggesting an acidification of the surface. Furthermore, by using coupling agents for immobilization of the bioactive mixture onto the surface determined a narrowing of the range between minimum and maximum

contact angles indicating that some groups from the bioactive mixture are involved in reaction with the functionalized substrate. The obtained stratified composites present pH responsiveness, switching between hydrophilic and hydrophobic surfaces at pH ≈ 6. Polyethylene is a well-known material used as implant biomaterial because of its proper mechanical properties and inertness [41, 42]. High hydrogen ion concentrations have been found in inflamed tissue (down to pH 5.4) in fracturerelated hematomas (down to pH 4.7), in cardiac ischemia (down to pH 5.7) and around malignant tumours [43]. Therefore, an implant material that has pH responsiveness in the acidic region (below the physiological pH) could be very useful when releasing bioactive substances is needed. Based on the critical pH of the responsive chitosan/vitamin E-modified polyethylene, it can be concluded that this material presents potential as active-biomedical implant.

Conclusion

Fig.  5  a Contact angle (θ) titration curves of the substrates and b derivative curves of dθ/dpH versus pH for chitosan/vitamin E-coated PE films

Stable dual bioactive coatings based on chitosan and vitamin E mixture were obtained on top of polyethylene substrate by applying a two-step procedure based on corona pre-activation and coupling reaction followed by electrospraying deposition. The vitamin E/chitosan composition determines the rheological properties such as viscosity decrease with increasing vitamin E content, change of the gel-like behavior to a fluid-like behavior, which further influenced the electrospraying process and morphology of the deposited coating. The chemical immobilization of chitosan/vitamin E mixture on corona treated PE surface was achieved using coupling agents, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) and carbonyldiimidazole (CDI), the first system being more effective in terms of grafting degree and stability of the coating. Chitosan and vitamin E interacts mainly electrostatic and through hydrogen bonds. The obtained materials retain their bulk properties (mechanical, thermal, a.s.o.) because corona treatment and deposited chitosan/vitamin E electrospun layer is very thin affecting only the surface properties. The functionalized surface shows pH responsiveness switching from hydrophilic to hydrophobic characteristic at pH ≈ 6. The chitosan/vitamin E-modified PE substrate inhibited the growth for three different bacterial strains (gram-negative and gram-positive) and presented antioxidative properties, acting as radical scavenging surfaces. The dual bioactive character maintained even after subjecting the samples to harsh desorption environment. The obtained material is of particular relevance for some fields like food packaging/ processing, pharmaceutical packaging, implant technology, and prosthetic devices.

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Acknowledgments  The authors acknowledge the financial support given by Romanian CNCS through the project BIONANOMED 164/2012, by International Atomic Energy Agency (IAEA) through research Project No. 17689/2013 and also by COST Action FA0904.

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