Lactoferrin-Immobilized Surfaces onto ... - ACS Publications

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Lactoferrin-Immobilized Surfaces onto Functionalized PLA Assisted by the Gamma-Rays and Nitrogen Plasma to Create Materials with Multifunctional Properties Elena Stoleru,† Traian Zaharescu,‡ Elena Gabriela Hitruc,† Alenka Vesel,§ Emil G. Ioanid,† Adina Coroaba,† Agnes Safrany,∥ Gina Pricope,⊥ Maria Lungu,# Christoph Schick,¶ and Cornelia Vasile*,† †

“P. Poni” Institute of Macromolecular Chemistry, Physical Chemistry Department, Iasi 700487, Romania National Institute for R&D in Electrical Engineering, Bucharest 030138, Romania § Jožef Stefan Institute, Ljubljana 1000, Slovenia ∥ Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna 1400, Austria ⊥ Veterinary and Food Safety Laboratory, Food Safety Department, Iasi 700487, Romania # National Institute of Research and Development for Biological Sciences, Bucharest 060031, Romania ¶ Universität Rostock, Institut für Physik, Rostock 18059, Germany ‡

S Supporting Information *

ABSTRACT: Both cold nitrogen radiofrequency plasma and gamma irradiation have been applied to activate and functionalize the polylactic acid (PLA) surface and the subsequent lactoferrin immobilization. Modified films were comparatively characterized with respect to the procedure of activation and also with unmodified sample by water contact angle measurements, mass loss, X-ray photoelectron spectroscopy (XPS), attenuated total reflectance-Fourier transform infrared spectroscopy (ATRFTIR), atomic force microscopy (AFM), and chemiluminescence measurements. All modified samples exhibit enhanced surface properties mainly those concerning biocompatibility, antimicrobial, and antioxidant properties, and furthermore, they are biodegradable and environmentally friendly. Lactoferrin deposited layer by covalent coupling using carbodiimide chemistry showed a good stability. It was found that the lactoferrin-modified PLA materials present significantly increased oxidative stability. Gammairradiated samples and lactoferrin-functionalized samples show higher antioxidant, antimicrobial, and cell proliferation activity than plasma-activated and lactoferrin-functionalized ones. The multifunctional materials thus obtained could find application as biomaterials or as bioactive packaging films. KEYWORDS: functionalized surface, lactoferrin, PLA, carbodiimide chemistry, plasma, gamma-rays

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INTRODUCTION

backbone ester groups, and the degradation rate depends on the PLA crystallinity, molecular weight, molecular weight distribution, morphology, water diffusion rate into the polymer, and the stereoisomeric content.6 Another important limitation of PLA is the lack of compatibility for cells. It is difficult to transplant isolated cells in PLA scaffold because cell attachment on PLA is rather low due to its hydrophobicity (static water contact angle of approximately 80°),7 and this results in low cell affinity and can elicit an inflammatory response from the living host upon direct contact with biological fluids.8,9 PLA is chemically inert with no reactive side-chain groups, making its surface and bulk modifications a challenging task.10 One

Poly(lactic acid) (PLA) has been receiving much attention in the last decades due to its biodegradability in the human body as well as in the soil, biocompatibility, environmentally friendly characteristics, and nontoxicity.1−3 Poly(lactic acid) (PLA) is a highly versatile biodegradable material derived from completely renewable resources (like corn, sugar beet, wheat, and other starch-rich products),4 and its applications were initially oriented toward the manufacture of medical grade sutures, implants, and controlled drug release applications mainly because of its high costs. Actually, PLA has many potential uses including medical and textile industries, as well as the packaging industry.5 PLA limitations consist in a poor toughness, high brittleness with less than 10% elongation at break, and slow degradation rate. It is known that PLA degrades through the hydrolysis of © XXXX American Chemical Society

Received: July 22, 2016 Accepted: October 27, 2016

A

DOI: 10.1021/acsami.6b09069 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

the formation of peroxyl structures, or grafting of other type of molecules.21 The degradation of the polymer is evidenced by reduction in molecular weight and activation energy of thermal degradation.22,23 The tremendous radiochemical features of PLA are offered by its high susceptibility to form free radicals, which further interact with other molecules. The polymer blends of PLA with collagen,24 polyethylene,25 or poly(vinyl acetate-co-vinyl alcohol)26 can be compatibilized by the bonding of PLA radicals on the backbones of the second component by using gamma irradiation. The new structures become biodegradable materials, extending their application areas to the food packaging sheets and medical wear products with reasonable long-term stability. The durability improvement of PLA products can be achieved by radiation cross-linking27 and/or using in situ compatibilization.21 Lactoferrin (LF) is a globular glycoprotein with multiple bioactive properties important in several physiological functions, such as immune response, antioxidant, regulation of iron absorption in the bowel, anticarcinogenic and antiinflammatory properties; furthermore, it offers protection against microbial infection. All the above-mentioned properties make it favorable for utilization in obtaining new bioactive materials with different characteristics for humans, animals, and materials with respect to their protection against microorganism attack.28 It is known that if a protein does not have the proper conformation, its activity is diminished or lost. The activity of LF, as is the case of most proteins, is highly dependent on the three-dimensional or tertiary structure of the protein, so the instability limits its usefulness. Microbial transglutaminase (mTGase) has been used to catalyze the immobilization of lactoferrin onto the wool fabrics, and the good antibacterial properties of immobilized wool against both Gram-negative and Gram-positive bacteria are demonstrated.29 This paper deals with lactoferrin immobilization onto the PLA substrate to obtain multifunctional biocompatible surfaces with antibacterial/antioxidant activities. This was achieved by using a two-step procedure consisting in cold plasma or gamma irradiation activation followed by a traditional wet-coating based on carbodiimide chemistry. As far as we know, the effects of ionizing radiation and cold plasma as pretreatments for PLA activation and functionalization and lactoferrin immobilization has not been reported. In this research, primarily surface modification but also in some cases bulk modification of PLA was carried out by both mentioned physical procedures to achieve multifunctional surface properties by lactoferrin grafting. A two-step procedure was used for lactoferrin immobilization onto the PLA surface that allowed the preservation of the biological activity of the protein as antibacterial, antioxidant, and biocompatibility tests have shown, and also, the deposited protein layer has a good stability.

approach to solve this problem is to immobilize a biocompatible layer on the surface of the polymer to improve cell−material interactions.11 There are either physical or chemical treatments for modification of polymer surfaces. Generally, chemical treatments use organic solvents and generate wastes, which could be potentially harmful to the environment. The physical treatments based on plasma or gamma irradiation technologies are in many cases preferred, because apart from being environmentally friendly, with optimum mild conditions, they selectively modify the topmost layers. Especially for plasma treatment, no changes in the bulk properties of the material result.12 This is why the controlled surface functionalization of a broad range of polymers is achieved by plasma treatment technologies which are efficient and commercially applied.13 Plasma treatment technologies are likely to be the most useful commercial techniques for controlled surface functionalization of a broad range of polymers. Plasma treatment has been used for different purposes to modify or improve the surface features of poly(lactic acid). Dielectric barrier discharge (DBD) operating at medium pressure in different atmospheres was explored to increase PLA’s surface wettability to promote interaction with fibroblasts.14 The influence of Ar plasma on the surface polarity, morphology, thickness of ablated polymer layer, chemical composition of the polymer surface, and the zeta potential of PLA was studied. Plasma treatment improves significantly the cell adhesion and proliferation of vascular smooth muscle cells (VSMCs) on the PLA.15 In recent years, protein or oligopeptide immobilization on polymer surfaces to improve biocompatibility is of much interest. Immobilization of some special biologically active molecules on synthetic materials is of critical importance because they can in principle elicit some specific, predictable, and controlled responses from the cells seeded on the materials.11 Xia et al.16 reported a threestep surface modification method, by which biomolecules, such as gelatin and chitosan, are covalently immobilized on the surface of plasma PLLA via −COOH groups introduced by acrylic acid grafting polymerization. Results from in vitro study using human umbilical vein endothelial cells have shown better cell affinity of both modified PLLA substrates in comparison with PLLA, by improving cell adhesion, spreading, and focal adhesion, as well as by promoting cell proliferation and complete endothelialization. Ionizing radiation is a well-known technique for modification of polymers, and it allows the formation of radicals, which react further with each other, with oxygen or with other coupling agents. The ionizing radiation is employed in the polymer area to induce several chemical processes such as polymerization, cross-linking, grafting, and degradation of particular polymer components.17 The result of competition between scission, cross-linking, or grafting depends on several factors, including the chemical structure of the polymer, the absorbed dose, the dose rate, and the processing environment.18 According to the Charlesby−Pinner relationship, the ratio between radiochemical yields of scission and cross-linking is the main criterion in the estimation of polymer radiation stability. PLA has a value of G(S)/G(X) placed between 7 and 10.5.19 This means that this polymer is extensively degraded by molecular scission. The first step in the degradation mechanism of PLA is related with the presence of the tertiary carbon atoms,20 whose proton can leave molecules. These radical positions can be involved in a different reaction: oxidation, by

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EXPERIMENTAL SECTION

Materials. The poly(lactic acid) used in this study was type 2002D from NatureWorks LLC, having a density of 1.24 g/cm3, melt flow index (MFI) of 5−7 g/(10 min) (at 210 °C/2.16 kg), and a content of 96% L-lactide and 4% isomer D. Weight-average molecular weight determined by GPC was 4475 kDa. The PLA films were obtained by hot pressing of pellets using a Carver press at 175 °C (2 min premelting and 2 min pressing at 240 bar) with a thickness of 0.3 mm ±0.05 mm. For plasma treatment/gamma irradiation and further protein immobilization, the films were cut into 2 cm × 2 cm pieces, B


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ACS Applied Materials & Interfaces and their mass was approximately 45 mg. After the film was rinsed with ethanol, ultrasonicated, and dried, the PLA film was plasma-treated/ gamma-irradiated. Surface modification of PLA was done using lactoferrin (LF) from bovine milk (Sigma-Aldrich, Germany). For covalent bonding of LF onto the PLA surface in LF solution, two coupling agents were used (i.e., EDC (1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide); both from SigmaAldrich). Preactivation Treatment. Plasma Treatment. Plasma was generated using the cold-plasma experimental setup (Supporting Information (SI) Figure S1). The polymeric film samples (4 in Figure S1) were exposed onto both sites at various time periods of 10, 20, and 30 min to high-frequency plasma generated in N2, which was created inside a glass reactor, using a 0.4 mbar (40 Pa) pressure. Inside the reactor were two electrodes of 18 × 21 cm placed at a distance of 6.5 cm (3 in Figure S1) connected to a source of high frequency (1.3 MHz) at a discharge power of 100 W (1 in Figure S1). A highfrequency generator assures a very weak discharge, so the intensity is below 20−30 mA. The sample is placed at equal distance between the two electrodes so that the potential gradient is very low and corresponds to the “Faraday” zone. The nitrogen flow rate was of 1.1 × 10−9 m3/s. The plasma treatment consisted of two steps. First, the PLA surfaces were degassed for 1 min at a pressure 0.05. the method used in the interpretation of the results is given in the refs 37, 38. Chemiluminescence (CL). investigations were achieved with LUMIPOL 3 unit (SAS, Bratislava, Slovakia) as nonisothermal dependencies of recorded intensity on temperature on film samples with small weights not exceeding 5 mg. The selected temperature range starts from room temperature and ended at 250 °C. The measured temperatures had a low error (±0.5 °C). Heating rates were 2, 3.5, 5, and 10 °C min−1. CL determinations were carried out in air under static conditions. The CL intensities values are normalized to sample mass for their reliable comparison. The preparation of samples and the measurement procedure were reported earlier.39 Antibacterial Tests. Antibacterial tests were performed using three bacteria strains, namely, Salmonella typhimurium, Listeria monocytogenes, and Escherichia coli, which are usually tested for in food products of both animal and nonanimal origin. The bacteria strains were purchased from American Type Culture Collection (Rockville, MD) and have been 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; SR EN ISO 7218-A1/2014: Microbiology of food and animal feeding stuffsGeneral requirements and guidance for microbiological examinations. Amendment 1. The detailed procedure for antibacterial tests is described in ref 40. It is important to mention that a log reduction of 10log col/g of 0.052 is applied for each experiment. Antioxidant Activity Evaluation. DPPH Assay. DPPH (1,1diphenyl-2-picryl-hydrazyl) free radical assay was used to determine the percentage of radical scavenging activity (RSA%). Initially, 50 mg of LF-coated PLA film was placed in a flask containing 5 mL of a DPPH methanol solution of 10−4 M that was kept in dark. The reaction between DPPH radical and an antioxidant compound that is a hydrogen donor leads to a reduced form of DPPH. The reduction process is accompanied by a change in color (from deep violet to light yellow), which is monitored by measuring, after 30 min, the absorbance at 517 nm with a UV−vis spectrophotometer (Cary 60UV−vis Spectrophotometer, U.S.A.). The radical scavenging activity (RSA) of the tested materials was calculated according to eq 1:

⎛ A sample ⎞ %RSA = 100 × ⎜1 − ⎟ Acontrol ⎠ ⎝

juice, such as UV absorbance, pH, and conductivity, were performed to test the antioxidant activity of the lactoferrin-modified PLA materials on a real food product. The experimental details have been described previously.40 Cytotoxicity Direct Contact Test. Mouse fibroblasts from NCTC clone L 929 cell live purchased from American Type Culture Collection (ATCC, U.S.A.) were used for cytotoxicity testing. Cytotoxicity of obtained materials was evaluated by direct contact method and is presented in detail in the study by Stoleru et al.35

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RESULTS AND DISCUSSION Wettability and Mass Loss Evaluation. The wettability was evaluated by water contact angle measurements and mass loss by high-precision gravimetric measurements. The wettability and mass loss of poly(lactic acid) films dependences on plasma exposure time period and gamma irradiation dose are presented in Figure 1 and Figure 2.

Figure 2. Variation of water contact angle with γ-irradiation dose of PLA.

The water contact angles of PLA surfaces decreases by increasing the N2 plasma exposure time (Figure 1), the surface wettability being enhanced by incorporating polar groups on the PLA surface. By increasing the plasma exposure time, a direct proportional mass loss is observed. This corresponds to an etching of a very thin removed layer with a thickness of about 1−1.2 μm. There was no significant difference between the contact angles and mass loss of PLA surfaces irradiated with different doses. As revealed from contact angle data, plasma

(1)

where Asample represents the absorbance of the sample solution, and Acontrol represents the absorbance of PPH solution without the addition of the LF coated-film.40 Sensorial Analysis and Evaluation of Physical Properties. Sensorial analysis and evaluation of physical properties of the apple D


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Figure 3. AFM 2D (A) and phase (B) images for neat PLA (a) and nitrogen-plasma-treated PLA surface at 10 (b) and 20 min (c) and gamma irradiated with different doses of 10 kGy (d) and 30 kGy (e).

Figure 4. AFM 2D (A) and phase (B) of N2-plasma-treated and gamma-irradiated PLA with immobilized lactoferrin onto surface.

with a higher surface roughness (Figure 3b,c). In the case of gamma irradiation, the PLA surface does not present the ellipsoidal formations (the transition morphology), meaning that this treatment affects to a much greater extent the PLA surface rather than plasma (Figure 3d,e). Different morphologies are observed when LF surface immobilization is achieved in the presence of EDC+NHS coupling agents than that observed on physically adsorbed samples (Figure 4). After lactoferrin immobilization, the polymer surface becomes smother. The protein molecules fill the gaps between the two “hills”, which leads to a more homogeneous surface. Chemical immobilization of the lactoferrin onto gammairradiated PLA (PLA/30kGy/EDC+NHS/LF) induces a roughness increase. Lactoferrin is a molecule with a bilobal globular shape with dimensions of 4.0 × 5.1 × 7.1 nm.42−44 The lactoferrin molecules seem to attach laterally each other, leading to a sort of two-dimensional network morphology (Figure 4). By definition, the “average roughness, Ra, and root mean square, Rms, are two physical scales describing the roughness degree of the sample. Surface skewness, Ssk, is a statistical parameter describing the asymmetry of average high distribution peaks in histogram. For a symmetrical distribution (Gauss), Ssk = 0. The value Ssk 0, the surface presents peaks. The

exposure determines a more significant functionalization of PLA surface with polar groups than gamma irradiation (Figure 2). Surface Morphology. The surface morphology of nitrogen plasma and γ-irradiated PLA samples were determined by AFM in tapping mode on a 1 × 1 μm2 area. The 2D and phase images are shown in Figure 3 and Figure 4. In surface characterization of the biomaterials, the phase imaging atomic force microscopy represents a powerful tool. The phase images obtained give more information than the 3D morphological images, which are able to reveal chemical variation and detailed surface properties. By phase imaging, it is possible to detect viscoelastic dissipated properties (chemicaldependent phase) of the surface and to show detailed information about the surface morphology (morphologicaldependent phase) than corresponding topographic imaging.41 As noticed by AFM study, the surface morphology changes and the PLA surface roughness increases by increasing the plasma treatment time or irradiation dose. Particularly, as apparent in Figure 3, it is noticed that the unmodified PLA surface does not reveal significant cavities and protrusions. Only 10 min of nitrogen plasma treatment creates on the PLA surface a significant number of ellipsoidal formations with an average width of 62 ± 3 nm and length of 107 ± 10 nm. An increase of the treatment time leads to a link-up between these formations, resulting in a brain-like appearance of PLA surface E


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Figure 5. Average roughness of N2-plasma-treated (a) and γ-irradiated (b) samples.

kurtosis coefficient, Ska is a statistical measure of the comparison of the measured profile and the Gaussian distribution characteristic of a perfectly random distribution of peak heights and valley depths. If Ska = 3 there is a Gaussian distribution. When Ska 3 pores prevail.”45,46 The surface skewness and coefficient of kurtosis values were determined to be dependent on the gamma-irradiation dose or nitrogen-plasma-treatment time and also by lactoferrin deposition onto substrate. Longer plasma treatment time/ higher irradiation doses and lactoferrin grafting lead to a porous-like surface where peaks prevail as Ska >3 (Table S1 in Supporting Information). Each AFM image was analyzed in terms of surface average roughness (Ra). The AFM data reveals that the average surface roughness increases with treatment time and irradiation dose (Figure 5), and hence, it can support the adhesion improvement. These data are corroborated with the wettability results in the case of plasma treatment. A “chemical” mechanism due to the PLA degradation processes may be responsible for the highly rough surface. As the PLA is a heterogeneous system composed of ordered crystalline and amorphous parts is a known fact. The structural integrity of the polymer system is ensured by the amorphous regions located between the crystalline ones that bind together.47,48 The effect of high-energy plasma or γ rays on the PLA surface is stronger on amorphous regions than on crystalline ones, the former being less stable. Thereby, highly rough surface of the polymer is created by short-time plasma treatment mainly because of the degradation of amorphous regions as it was also found from DSC data. Knowing that gamma irradiation modifies both the bulk and surface properties of polymers was to be expected that its action on the PLA substrate will lead to a higher roughness. On the basis of wettability, weight loss, and morphology examination by AFM data, it was concluded that 20 min of plasma activation and a 30 kGy irradiation dose are the optimum parameters of PLA functionalization for lactoferrin immobilization. The additional analytical results will be presented only for the samples modified using the selected experimental conditions, as the data to be easier to follow. DSC Analysis. DSC analysis was performed for better explaining the morphology of gamma-irradiated and plasmatreated samples and to answer if the observed grains and protrusions on AFM images can be attributed to the crystalline phase of PLA.

In the DSC curve of native poly(lactic acid) (Figure 6) are noticed two endothermic peaks which are assigned to glass

Figure 6. DSC curves of plasma-treated or gamma-irradiated and lactoferrin-modified PLA samples.

transition and melting. At this heating rate, the cold crystallization is slightly observed for PLA. The glass-transition temperature (Tg) of PLA was 62 °C. Melting endotherm in PLA DSC curve, occurring at 153.9 °C, confirms its semicrystalline nature. No significant influence was observed of irradiation exposure and plasma treatment on the glass transition temperature (Tg) of poly(lactic acid) (PLA) (Table S2 in SI). The Tg values of the activated and modified samples varied from 61.7 to 62.99 °C with a little high values for lactoferrin-immobilized surfaces. The plasma affects only surface morphology of PLA, whereas gamma irradiation affects both surface and bulk morphology. This is evidenced mainly by changes in cold and premelting crystallization processes of PLA evidenced in DSC data (Table S2). The cold crystallization temperature (Tcc) of PLA (Table S2) gradually decreased by gamma irradiation/plasma treatment and lactoferrin grafting from 122.6 °C for neat PLA to 113.2 °C for PLA/30kGy/EDC+NHS/LF, which suggests these treatments enhance the ability to cold crystallization of PLA sample. The values of melting enthalpy (ΔHm) and cold-crystallization enthalpy (ΔHcc) increase by gamma irradiation and nitrogen plasma treatment (from 4.2 and 2.3 mJ/mg for neat PLA, to ΔHm = 20−23 mJ/mg or ΔHcc =13−14 mJ/mg, respectively), F


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more suitable for anchoring the protein onto PLA surface than gamma-irradiation. The ATR-FTIR spectrum of the native PLA and that of the nitrogen-plasma-activated (i.e., which is treated only with EDC +NHS and immersed in lactoferrin solution without coupling system after washing step with PBS and surfactant Tween 20) are similar, and therefore, the lactoferrin was totally removed from the substrate surface when it is physically adsorbed. The same conclusion was drawn from XPS spectra examination of the same samples. XPS Analysis. XPS as a surface-sensitive technique was used to characterize the native, nitrogen plasma, or γ-irradiated and lactoferrin-coated PLA surfaces in terms of chemical composition. The surface composition is an average of two measurements on different spots on the sample surface with a standard deviation of ±0.5 at. %. In Table 1 are listed the chemical elements and the corresponding atomic ratios found in the case of native, nitrogen-plasma-treated or γ-irradiated, and lactoferrin-modified PLA surfaces. After PLA substrate exposure to nitrogen plasma, in the XPS survey spectra there is an emission signal assigned to nitrogen atoms, which does not appear in the native PLA spectra. After lactoferrin coating, the nitrogen atomic percentage increases even more, the highest content being determined for the sample obtained by lactoferrin immobilization onto the PLA surface using coupling agents (EDC+NHS), which suggests that a better protein-functionalization of the PLA substrate is achieved. It seems like that the chemical immobilization is more efficient than the simple physical adsorption. A slight decrease of the oxygen atomic percentage by increasing the γ-irradiation dose was observed also. The C 1s high-resolution spectrum of native PLA has three peaks belonging to C−C, C−O, and OC−O groups. After plasma treatment, the intensity of the peaks assigned to C−O and OC−O groups decreased (also oxygen concentration is lower for this sample (Table 1). The shape of the C1s high-resolution spectrum of lactoferrincoated PLA surfaces (Figure 8) is typical for a protein, showing the presence of peptide and amino groups. However, the peaks assigned to carbon bond to oxygen (C−O and OC−O) from the virgin substrate are no longer visible. This means that the protein covers the whole surface. The C 1s high resolution spectra of lactoferrin-modified PLA surface can be curve-fitted with four peak components centered at 285.0 eV (C1), 285.9 eV (C2), 287.0 eV (C3), and 289.1 eV (C4). C1 corresponds to C-C and C-H bonds characteristic to the aliphatic part of the protein, C2 to C-NH2, C3 is assigned to C-O bonds, C4 is associated with the carbon atom involved in CO from HN-CO or COOR functional groups. One of the ways to achieve LF covalently bound onto plasma-treated or gamma-irradiated PLA surface is by amide

while by lactoferrin grafting, a decrease is observed Table S2. Even if the value of melting enthalpy (ΔHm) increases by gamma irradiation, a slight decrease is observed with increasing the irradiation dose (from 23 mJ/mg at 10 kGy to 18 mJ/mg at 30 kGy), which may due to the fact that at higher doses the crystalline regions are also affected. The increase of melting enthalpy (ΔHm) (which is direct proportional with the polymer crystallinity) is explained by the fact that amorphous regions are more easily accessible to degradation during the irradiation process. An increase in melting enthalpy during the γ radiation exposure is due to liberation of macromolecular fragments, which will exhibit higher mobility and can reorganize themselves leading to an increased crystallinity degree, according to the results reported by Zaidi et al.49 Moreover, before gamma irradiation, the DSC curve of PLA exhibits the presence of a single peak for the melting temperature at 154 °C. After gamma irradiation, it displays a shoulder on melting peak at about 149 °C, which may have different causes (Figure 6). It may be caused by reorganization during irradiation and/or occurrence of different crystal populations. This shoulder is not observed after plasma treatment but is evident in the DSC curve of gamma-irradiated and lactoferrin-grafted sample. ATR-FTIR Results. The ATR-FTIR spectra (Figure 7) confirmed the presence of lactoferrin onto PLA plasma-treated

Figure 7. ATR-FTIR spectra of plasma treated and γ-irradiated PLA surface modified with lactoferrin.

sample, especially when the immobilization was done by covalent coupling and not by physical adsorption. The absorption bands specific to lactoferrin and its covalent bonding with PLA functionalized surface (Amide I at 1645 cm−1, Amide II band at 1540 cm−1, and at 3400 cm−1 the O−H and N−H stretching vibrations) were more evident for N2plasma-pretreated PLA, meaning that this functionalization is

Table 1. Surface Composition of Native, Plasma-Treated or γ-Irradiated, and Lactoferrin-Coated PLA Samples sample PLA PLA/N2 PLA/N2 20 min/LF PLA/N2 20 min/EDC+NHS/LF PLA/30kGy PLA/30kGy/LF PLA/30kGy/EDC+NHS/LF

C [at.%] 61.9 62.7 65.8 67.7 61.3 59.6 62.8

± ± ± ± ± ± ±

O [at.%]

0.31 0.313 0.33 0.34 0.30 0.29 0.31 G

38.1 35.3 26.0 21.6 36.6 35.5 25.8

± ± ± ± ± ± ±

0.19 0.17 0.13 0.24 0.18 0.18 0.13

N [at.%] 2.0 8.2 9.7 1.2 2.1 5.7

± ± ± ± ± ±

0.01 0.04 0.05 0.006 0.01 0.03


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Figure 8. High-resolution C 1s spectra of nitrogen-plasma-treated or gamma-irradiated and LF-modified PLA surfaces. For each peak resulted after deconvolution of high-resolution C1s spectra is assigned a functional group in which the specific carbon atom is involved, as indicated by the arrows on the figures.

bond formation between surfaces containing oxygen functionalities (gamma-irradiated PLA) or amino groups (plasmatreated PLA) and lactoferrin amino or carboxylic groups by

means of using coupling agents EDC+NHS. The XPS atomic ratios C4/C2 (N-CO/C-NH2) gives information about amide bond formation between the functionalized PLA surface H


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Figure 9. (A) Nonisothermal CL spectra recorded on γ-irradiated PLA and modified with lactoferrin and (B) the onset oxidation temperatures (OOT) for pristine PLA (white) and lactoferrin-modified PLA (black) at different irradiation doses.

Table 2. OOT and Activation Energy Values Ea for Processed PLA and Lactoferrin-Modified PLAb OOT (°C) sample

dose (kGy)

2

PLA

0

203

10

200

20

195

30

190

10

212

20

215

30

218

10

221

20

224

30

227

PLA/LF

PLA/EDC-NHS/LF

PLA/N2 20 min

202

PLA/N2 20 min/LF

214

PLA/N2 20 min/EDC+NHS/LF

224

3.7

5

214 228 Y = −7.21 + 8958.5X (0.98008/Ea = 74.48 kJ mol−1)b 208 225 Y = −8.31 + 9358.3X (0.97773/Ea = 77.67 kJ mol−1) 208 221 Y = −6.89 + 7843.1X (0.97486/Ea = 65.17 kJ mol−1) 205 218 Y = −6.48 + 7631.3X (0.96292/Ea = 63.47 kJ mol−1) 225 234 Y = −7.48 + 9108.1X (0.98207/Ea = 74.05 kJ mol−1) 228 238 Y = −7.44 + 9126.6X (0.98136/75.75 kJ mol−1) 228 242 Y = −7.44 + 9126.6X (0.98136/Ea = 75.75 kJ mol−1) 228 239 Y = −15.75 + 13540.1 X (0.9236/Ea = 112.38 kJ mol−1) 232 240 Y = −18.55 + 15040.0X (0.97268/Ea = 124.83 kJ mol−1) 233 243 Y = −19.56 + 15617.3X (0.91486/Ea = 129.63 kJ mol−1) 213 226 Y = −6.16 + 8428.7X (0.98878/Ea = 69.95 kJ mol−1) 222 232 Y = −12.73 + 11862.9X (0.9638/Ea = 98.46 kJ mol−1) 233 240 Y = −20.06 + 15807.7X (0.97397/ Ea = 131.20 kJ mol−1)

10 240 235 230 228 245 247 250 247 249 250 242 244 248

a Y and X correspond to the eq 1; in brackets are given correlation coefficient and average activation energy. bThe error in OOT values determination is ±2 °C, and the average Ea is ±5 kJ mol−1.

Chemiluminescence Results. Poly(lactic acid) sheets were in the first step γ-irradiated or subjected to plasma treatment in N2 environment, when free radicals are formed. These two energetic processes create reactive spots to which either oxygen molecules can be attached and peroxyl structures are created40 or nitrogen-containing functionalities to which lactoferrin can be attached. The increase of CL emission intensities with temperature is the oxidation evidence of radical fragments. The smooth enhancement of signal intensities over the low and medium temperature ranges suggests the slow decay of residual radicals after pretreatment operations. The full oxidation starts after 150 °C (Figure 9A), when the molecules

and lactoferrin. The C4/C2 atomic ratio of all lactoferrinmodified plasma treated/gamma irradiated PLA samples increases after using coupling agents EDC+NHS from 0.45 corresponding to PLA/N2/LF to 0.68 for PLA/N2/EDC +NHS/LF, which proves the covalent bonding of lactoferrin. Gamma irradiation determines functionalization of PLA substrate by oxygen and nitrogen-containing group incorporation (Table 1 and Figure 8). By comparing the two functionalization methods, it can be observed that the incorporation of nitrogen functional groups at the PLA surface is higher for plasma exposure than in γirradiation. I


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Table 3. Antibacterial Activity of Untreated, Plasma, and/or Gamma-Irradiated PLA and Modified with Lactoferrin Escherichia coli inhibition (%)

Listeria monocytogenes inhibition (%)

Salmonella typhimurium inhibition (%)

samples

24 h

48 h

24 h

48 h

24 h

48 h

PLA PLA/N2 20 min/EDC+NHS/LF PLA/30KGy/EDC+NHS/LF

35 ± 0.88 44 ± 1.10 72 ± 1.80

52 ± 1.30 75 ± 1.87 100 ± 2.53

31 ± 0.93 46 ± 1.38 100 ± 3.11

40 ± 1.20 71 ± 2.13 100 ± 3.28

43 ± 0.86 70 ± 1.40 100 ± 2.03

55 ± 1.11 87 ± 1.74 100 ± 2.09

provides a high efficiency of grafting onto plasma-treated PLA (Table 2 and Figure S2). The start of oxidation is extensively delayed. The onset oxidation temperatures of PLA/N2 20 min/ EDC+NHS/LF become higher because the radical positions are effectively blocked. The thermal stability of γ-irradiated PLA is characterized by lower activation energies. However, after lactoferrin grafting, its stability is improved accordingly with the nature of reacted moieties. CL curves describes the presence of peroxides on the same temperature range as for lactoferrin-modified PLA. However, the measured amount is lower, which is the result of their decay during grafting. The thermal stability of grafted PLA is higher than the pristine material, as revealed through the comparison of activation energies of thermo-oxidative degradation (from 58.53 kJ mol−1 for PLA to 121.97 kJ mol−1 for PLA/10kGy/EDC+NHS/LF). The oxidation onset temperature increases as expected with heating rate in all cases, decreases with increasing gamma irradiation dose, and for each dose increases for lactoferrin-modified samples with respect to neat PLA. A similar variation after modification is also remarked in the values of activation energy of oxidative degradation, indicating an increase of thermo-oxidative stability after surface modification; therefore, on the basis of these results, it can be concluded that the lactoferrin is a good antioxidant to protect the surface of PLA material. The oxidative stability of lactoferrin-modified PLA is determined by the presence of aromatic amino-acids residues in protein primary structure, such as tryptophan.51 Antibacterial Tests. The obtained samples were tested for the growth inhibition of three different bacteria Escherichia coli, Listeria monocytogenes, and Salmonella typhimurium. As observed from Table 3, the modification of the surface layer of the PLA film with the nitrogen plasma and γ radiation methods resulted in sterilization of the film. Gamma ray doses used for sterilization purposes are usually less than 20 kGy.26 Bacteria growth was significantly inhibited from 24 to 48 h post incubation using lactoferrin-modified PLA samples from 40− 55% to 70−100%. Interaction between the lactoferrin and bacteria is modulated by a complex series of interactions. In general the antibacterial effects of lactoferrin are determined by the interactions which involve iron sequestration, being considered the primary bacteriostatic activity, because iron-free lactoferrin was more effective at inhibiting bacterial growth than iron-loaded lactoferrin, but it was also shown to be associated with an Nterminal peptide fragment that is released from lactoferrin by proteolysis.52 Besides the direct effect on the viability and growth of bacteria, the lactoferrin is capable of interfering with processes that involve bacterial surface components through a variety of mechanisms that are gradually being unearthed.52 The lactoferrin binds avidly to lipopolysaccharides (LPS) from Gram-negative bacteria53 and is capable of modulating the host response to LPS.54,55

are split and oxidized. The values of onset oxidation temperatures (OOT) measured on the pristine PLA functionalized at increasing irradiation doses or by plasma exposure do not significantly differ. The bonding of lactoferrin on PLA backbones occupies the reactive positions sensitive for oxygen attack. The efficiency of lactoferrin coupling on irradiated PLA is proved by the delaying start of oxidation, because the grafted lactoferrin offers an improved thermal stability to polymer support molecules. The γ-irradiated PLA samples display a shift of OOT to higher values as the dose in longer exposed samples (Figure 9B) according to the corresponding concentrations of radiolytically formed radicals. During the chemiluminescence measurements, the greater amounts of lactoferrin prevent oxidation. Even the grafted lactoferrin is susceptible to thermal oxidation, and the amounts of excited carbonyl structures which emit CL photons by the dropping down on the fundamental level are related only to the degradation of polymer chains. Consequently, the degradation behavior of PLA-LF samples is restricted to the oxidation damage of polymer matrix. The gamma-irradiation pretreatment of PLA sheets is an energetic processing for the modification induced in polymer substrates.49 Because there is an efficient energy transfer from the incidental ray onto the molecules, the local concentrations of free radicals that appeared during exposure are great. The grafting of PLA with lactoferrin is more intensive in comparison with the effect of γ-irradiation, where the transferred energy is notably lower. The amount of the photoresponsive intermediates accumulated during plasma action give a slightly improved stability with respect to the γirradiated PLA samples. The overall activation energy of thermo-oxidative degradation was evaluated by Kissinger’s method,50 which is based on the following equation: ⎛ β⎞ ⎛ A·R ⎞ E ln⎜⎜ 2 ⎟⎟ = ln⎜ ⎟− a ·Tp E R ⎝ a ⎠ ⎝ Tp ⎠

(2)

where β is the heating rate, Tp is OOT (onset oxidation temperature [K]), A pre-exponential factor (min−1), R is gas constant (8.314 × 103 kJ mol−1 K−1) and Ea is the overall activation energy (kJ mol−1). The values are given in Table 2. The average activation energy required for the thermal oxidation of plasma-treated PLA is 81.99 kJ mol−1, while the γradiolysed PLA needs maximum 77.67 kJ mol−1 at 10 kGy (Table 2). The presence of lactoferrin in the PLA-LF samples causes the increase in the activation energy from about 74 kJ mol−1, calculated for γ-exposed sheets, to 98.46 kJ mol−1 evaluated for plasma irradiation of similar samples. The addition of coupling agents in the immobilization process of lactoferrin increases the thermal stability of modified PLA (Table 2). This effect can be correlated not only to the efficiency of reagents on the jointing of lactoferrin, but also to the lack of peroxyl intermediates, which exist after the γirradiation. They initiate oxidation amplifying the material susceptibility to the oxygen attack. The pair of coupling agents J


Research Article

ACS Applied Materials & Interfaces The covalent attachment of lactoferrin to polymer surfaces can render the modified surface capable of killing bacteria on contact. This method does not involve the release of antisepticsthe lactoferrin molecules intrinsically destroy bacteria upon contact, by virtue of its structure. By directly disrupting the cell walls, this treatment avoids the biochemical route which often leads to resistance.56 The sample obtained by gamma preirradiation of PLA substrate and lactoferrin surface immobilization is the most antibacterial active. Antioxidative Activity Evaluation. Evaluating the free radical scavenging activity by DPPH assay was established that the sample obtained by lactoferrin grafting imparts antioxidant activity to PLA substrate (RSA 30%). In the case of lactoferrinmodified PLA substrate, the gamma-preirradiated sample has more pronounced antioxidant activity RSA of 65% (Table 4) when compared with the nitrogen-pretreated sample, even though the chemical grafting was more significant in the last case (as FTIR and XPS analyses showed).

Enzymatic browning of apples (fruits) is a process caused by the oxidation of phenolic compounds into quinones.57 Apple juice was subjected to enzymatic browning in the presence of the selected antibrowning agents prepared in this study, namely, nitrogen-plasma-treated and gamma-irradiated PLA modified with lactoferrin. By monitoring the color, pH, and conductivity of the apple juice, the relative effectiveness of these antibrowning agents for inhibition of enzymatic the browning of the juice was determined during 3 days of storage. The inhibition of polyphenol oxidase activity was assayed by measuring the rate of increase in absorbance at 450 nm. Higher values for absorbance at 450 nm correspond to higher browning/ darkening of the juice (see Figure 10).58 In presence of the gamma irradiation and lactoferrinmodified PLA, the apple juice kept its initial general aspects and properties a longer period of time. The change in color or browning do not appear after 48 h, whereas for other samples (native PLA and only gamma irradiated) , the changes appear after 24 h or less (2 h for juice placed on PET as commercial packaging material). Correlation of these results with RSA indicates that lactoferrin immobilization improved antioxidant activity of PLA film. It is well-known that the biomaterials attached to surfaces raise the question about their stability and durability of their effect.59 The stability of the lactoferrin-modified PLA films against air under ambient conditions was monitored over time. These samples were left in air under ambient conditions for 2 months. The surface wettability of these lactoferrin-modified samples remained unchanged over a period of more than 2

Table 4. Radical Scavenging Activity (RSA) of Untreated, Plasma, and/or γ-Irradiated PLA Substrate Further Modified with Lactoferrin sample PLA PLA/N2 20 min PLA/N2 20 min/EDC+NHS/LF PLA/30kGy PLA/30kGy/EDC+NHS/LF

RSA (%) 0 8 30 11 65

± ± ± ±

1.2 2.1 1.3 2.3

Figure 10. Variation in time of the absorbance at 450 nm (a), pH (b), and conductivity (c) of the apple juice in the presence of lactoferrin-PLA substrates obtained by either plasma or γ radiation activation and lactoferrin immobilization in comparison with apple juice sprayed onto commercial polyethyleneterephthalate film. K


Research Article


lactoferrin offers improved thermoxidative stability to polymer support molecules. On the basis of chemiluminescence results, it can be concluded that the lactoferrin-deposited layer is a good antioxidant which protects the surface of PLA material. The chemical grafting converts PLA into an upgraded material, which exhibits a convenient thermal stability and a useful functionalization. The covalent attachment of lactoferrin to the PLA substrate rendered a modified surface capable of killing bacteria on contact and a material with antioxidant properties. The obtained samples are not cytotoxic, and they stimulated cellular division, especially those obtained by the gamma irradiation activation. Gamma-irradiated activated samples and with immobilized lactoferrin show higher antioxidant, antimicrobial, and cell proliferation activities than that of plasmaactivated and lactoferrin-functionalized. These multifunctional materials are promising candidates for application as biomaterials or as bioactive packaging films.

months. Also, the samples were tested for their antibacterial and antioxidant activity immediately after preparation and after 2 months, and no significant differences were found between the antioxidant activities or bacterial inhibition capacities. It can be concluded that the lactoferrin-modified PLA materials kept their properties for a long period of time. Cytocompatibility In Vitro Testing. The in vitro cytotoxicity of lactoferrin-modified PLA materials was evaluated on the basis of cell proliferation, viability, and morphology. Fibroblast primary cultures obtained from mouse explants were used. To analyze possible release of toxic products by the tested materials, mouse fibroblasts were grown in contact with polymeric samples (Table 5). Table 5. Cell Viability on Native PLA and LactoferrinSurface-Modified PLA Samples by Plasma Activation and Gamma Irradiation after 24 and 48 ha

■

cell viability (%) sample PLA PLA/N2 20 min PLA/N2 20 min/EDC+NHS/LF PLA/30 kGy PLA/30 kGy/EDC+NHS/LF a

24 h 95.51 94.55 97.44 93.91 102.24

± ± ± ± ±

1.43 1.42 1.46 1.40 1.53

95.66 97.59 99.16 104.10 106.27

± ± ± ± ±

ASSOCIATED CONTENT

* Supporting Information

48 h

S

1.43 1.46 1.49 1.56 1.59

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b09069. (PDF)

■

Three measurements for each determination were done.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

The PLA sample did not have any influence on the fibroblast culture, which is based on its inert character. All other studied samples have a cellular viability of almost 100% or over with an increase of 4−6% for gamma preirradiated and LF-modified ones. The highest mouse fibroblast cells density was obtained for PLA/30 kGy/EDC+NHS/LF sample (Figure S3). It is well-known that cell morphology represents an important factor for any cellular function, such as proliferation, migration, and biosynthetic activity. In all experiments, the reference sample was represented by mouse fibroblasts cultivated in the absence of polymer specimens on TCPS (Tissue Culture Polystyrene). The results of morphological characterization of mouse fibroblasts grown on surface of polymeric samples (after 48 h of cultivation) were observed as light microscopy images and image processing (Figure S3). Light microscopy images reveal a normal phenotype of the cells presenting euchromatic nuclei. Therefore, all these samples are non-cytotoxic samples; the cells are normal from morphological point of view and only a few cells are altered and have smaller dimensions. Summary and Conclusions. The poly(lactic acid) surface was successfully functionalized by N2 cold plasma or gamma irradiation and further anchoring the lactoferrin (LF) onto the surface. The PLA surface functionalization conditions were optimized with respect to the variation of plasma exposure period and the gamma irradiation dose. On the basis of wettability, weight loss and AFM data was established that 20 min of plasma treatment and 30 kGy irradiation dose are the optimum parameters for PLA functionalization. Then, by using carbodiimide chemistry, the formation of covalently bonded lactoferrin-immobilized surfaces is ensured. ATR-FTIR and XPS analyses reveal that plasma pretreatment leads to a better lactoferrin grafting onto the PLA surface. The efficiency of lactoferrin coupling on plasma or gamma-irradiated PLA is proved by the delayed start of oxidation, because the graft

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the Romanian UEFISCDI through the research project BIONANOMED 164/2012 and IAEAVienna, Austria for the research project No. 17689/2013. The authors thank to Dr. Andreas Wurm, University of RostockGermany for technical assistance in DSC recordings.

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