ISSN 00181439, High Energy Chemistry, 2012, Vol. 46, No. 1, pp. 60–64. © Pleiades Publishing, Ltd., 2012. Original Russian Text © T.S. Demina, M.Yu. Yablokov, A.B. Gil’man, T.A. Akopova, A.N. Zelenetskii, 2012, published in Khimiya Vysokikh Energii, 2012, Vol. 46, No. 1, pp. 64–69.
PLASMA CHEMISTRY
Effect of DirectCurrent Discharge Treatment on the Surface Properties of Chitosan–Poly(L,LLactide)–Gelatin Composite Films T. S. Demina, M. Yu. Yablokov, A. B. Gil’man, T. A. Akopova, and A. N. Zelenetskii Enikolopov Institute of Synthetic Polymer Materials, Russian Academy of Sciences, ul. Profsoyuznaya 70, Moscow, 117393 Russia Email:
[email protected] Received June 15, 2011; in final form, June 27, 2011
Abstract—The surface of films made from a chitosan–poly(L,Llactide)–gelatin mixture stabilized with a graftedcopolymer fraction has been modified by dc discharge treatment, as well as that of films made of the individual components. The surface properties of the films (wettability, surface energy), the chemical struc ture of surface layers, and their morphology have been examined by goniometric measurement of contact angles, Xray photoelectron spectroscopy, and scanning electron microscopy. DOI: 10.1134/S0018143912010110
it possible, first, to abandon toxic solvents and, sec ond, to effectively combine the initial components with a high yield of the graft copolymer [5, 6]. Modification of the surface properties of polymers used in medicine and biotechnology with the aim of improving their hydrophilicity, cell adhesion, and polymer–tissue interaction is receiving progressively increasing attention from researchers since recently [7–9]. Lowtemperature, lowpressure plasma is used for treatment, which generate ions, radicals, free elec trons, and excited molecules inducing the processes of oxidation, crosslinking, formation of new polar groups, etc. The efficacy of modification depends on the treat ment conditions and the chemical structure of the poly mer surface [10, 11]. Various properties, such as adhe sion, permeability, hydrophilicity, and biocompatibil ity, can be changed by the treatment, with the bulk properties of the material remaining intact. [10, 12]. In this study, we decided to combine both approaches to PLLA modification and to examine the effect of lowtemperature plasma treatment on the surface properties of materials based on the chito san/PLLA/gelatin blend stabilized by the graft copol ymer fraction.
Biocompatible and biodegradable poly(L,Llac tide) (PLLA) is widely used in various fields of medi cine and biotechnology to make shafts, prostheses, suture filaments, and implants for bone and soft tissues and for targeted drug delivery. However, because of surface hydrophobicity, its use as a matrix for tissue engineering and cell carriers is substantially limited by low cell adhesion. One of the ways of resolving this problem is to modify PLLA by preparing graft copolymers and blends with natural polymers [1]. Materials based on PLLA modified in this way exhibit enhanced biocom patibility and cell adhesion and increase cell prolifera tion, facilitating the regeneration processes [2]. The most promising polymer in this respect is chitosan. Owing to its biodegradability, nontoxicity, and bacteri cidal and antimicrobial properties, this polysaccharide is an excellent material for use in tissue engineering, and the presence of positive charge on the amino group at neural pH values ensures high bioadhesive ness [3]. The addition of gelatin to the system improves the biocompatibility of the material. However, the synthesis of copolymers based on chi tosan and polylactide is fraught with difficulties. When the process is conducted in a polyester melt, chemical interaction between components is insignificant and proceeds only on the surface of the polysaccharide component because of low compatibility of the com ponents and chitosan infusibility. The mixtures obtained in this way are a composite in which the polysaccharide is distributed unevenly, retaining the initial particle size [4]. Running the process in a solu tion requires the use of expensive and highly toxic sol vents. The solidstate synthesis of such systems makes
EXPERIMENTAL Chitosan—poly[(1 → 4)2amino2deoxyβD glucose]—prepared by solidstate synthesis from crab shell chitin [13] was used. The chitosan molecular mass was 60 kDa and the degree of acetylation was 0.1 according to potentiometric titration and elemental analysis data. Poly(L,Llactide), a semicrystalline polyester with a molecular mass of 160 kDa and 60
EFFECT OF DIRECTCURRENT DISCHARGE TREATMENT
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Table 1. Effect of plasma treatment on the surface characteristics of test samples θ, deg
Sample CPG
PLLA
Chitosan
Gelatin
initial on cathode treated on anode initial on cathode treated on anode initial on cathode treated on anode initial on cathode treated on anode
of water
of glycerol
of water
of glycerol
γ
γp
γd
76 22 10 75 11 12 68 32 24 63 21 13
73 66 36 71 56 37 61 24 10 69 42 30
90.4 140.3 144.5 91.6 144.3 144.0 100.1 134.5 139.3 105.9 140.8 143.7
79.8 89.2 114.7 84.0 98.9 114.0 94.1 121.3 125.8 86.1 110.5 118.3
28.1 131.1 81.9 29.0 115.3 81.7 35.4 62.2 66.8 42.0 79.4 76.0
20.8 126.4 78.0 18.0 114.5 78.1 19.0 41.9 44.7 38.7 76.5 68.6
7.3 4.7 3.9 11.0 0.8 3.6 16.4 20.3 22.1 3.3 2.9 7.4
mp 165°C, was used as purchased from Sigma without further purification. Food gelatin of the P9 brand was also uses without additional purification. The chitosan—PLLA–gelatin (CPG) blend stabi lized by the graftcopolymer fraction was obtained via solidstate synthesis [14] by the action of pressure and shear stress in a Berstorff ZE40 doublescrew extruder at a component mass ratio of 52 : 13 : 35. The initial mixture was first homogenized at 50°С and a screw rotation speed of 100 rpm; the synthesis was run at the reactivemixing temperature of 100°С (below the components’ melting temperatures). The films were formed from a stable colloidal suspension of the mixture (5 wt % polymer) in dichloromethane by cast ing onto a glass substrate. It was impossible to shape a model threecomponent film without preliminary chemical modification of the mixture. Therefore, in order to determine the contribution made by each component to the behavior of the blended composi tion, films from the individual initial components were formed and studied. The film samples of unmodified polymers were prepared by dry forming from 5% chi tosan solutions in 4% acetic acid, gelatin in deionized water, and PPLA in dichloromethane. The films were dried at ambient temperature under equilibrium con ditions until complete dryness (~7 days); the thickness of the ready films was ~100 µm. The films were modified in lowpressure direct current discharge according to the procedure described in [15]. A copolymer film was placed on the anode or cathode and treated at a discharge current of 50 mA for 50 s, using residual air at a pressure of 13.3 Pa as the working gas. The surface properties were characterized by values of the contact angle (θ) measured with an Easy Drop DSA100 instrument (KRUSS, Germany) and Drop Shape Analysis V.1.90.0.14 software, using two test liq HIGH ENERGY CHEMISTRY
γ, mJ/m2
Wa, mJ/m2
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uids, (deionized) water and glycerol. From the exper imental values of θ, the work of adhesion (Wa), the total surface energy (γ), and its polar (γр) and disper sion (γd) terms were calculated according to the proce dure described in [16]. The chemical structure of the surface of the initial and modified films was studied by Xray photoelectron spectroscopy (XPS). XPS spectra were measured on a Riber LAS3000 instrument equipped with an OPX150 hemispherical retardingfield analyzer. Photoelec trons were excited by Xrays from an aluminum anode (AlKα = 1486.6 eV) at a tube voltage of 12 kV and an emission current of 20 mA. Peak positions were cali brated with reference to the standard С1S peak (284.6 eV) [17, 18]. The atomic concentrations of ele ments were calculated by conventional equations using photoelectron peak areas and the elemental response factors borrowed from [19]. Microphotographs of the surface were obtained with a Jeol JSM5300LV scanning electron micro scope at a voltage of 20 kV and a magnification of 1000. RESULTS AND DISCUSSION Plasma treatment results in a substantial decrease in the contact angle for both composite films and film samples of the individual components (Table 1). These data agree with published data and the results obtained for initial chitosan and PLLA modified in various gas plasmas [7, 20–22]. The contact angles determined with the two test liquids for the initial sample of the blended composi tion correspond to the values of θ obtained for the untreated PLLA film, thereby indicating that the sur face layer is enriched in polylactide chains. This can be due to the procedure of the formation of film samples
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Table 2. Chemical composition of surface layers of chito san–PLLA–gelatin films Atomic concentration, % Sample Untreated film Treated on cathode Treated on anode
C
O
N
54.1 60.8 74.8
45.9 34.4 21.9
– 4.8 3.3
Table 3. Binding energies found for CPG films Sample
C1S
O1S
N1S
eV Untreated CPG film Anodetreated CPG film Cathodetreat ed CPG film
283.7 285.5 288.5 284.9 286.8 288.3 285.0 288.0 289.4
532.3 (100%)
531.5 (100%) 400.1 (100%) 402.0 532.5 (100%) 407.0
from the chitosan/PLLA/gelatin blend, which forms in organic solvents stable colloid micellar dispersions with an average aggregate size of 400–800 nm accord ing to dynamic light scattering data. According to the 1H NMR data [6], aggregates of a chitosan–PLLA copolymer in CDCl3 prepared with out addition of gelatin to the reaction mixture, have the core–shell structure. The main proton peaks of the spectra correlate with the signals of polylactide chains, but they are resolved not so well as in the spectra of PLLA. Thus, polysaccharide units compose the core of nanomicrodispersions in organic solvents and grafted polylactide chains form the stabilizing shell. Consequently, when the film is formed from an organic solvent, the surface layer is enriched in PLLA homopolymers and grafted polylactide side chains. The films obtained with the use of gelatin admixture seem to have a similar structure. The treatment of CPG films on the cathode or anode leads to a considerable decrease in the contact angle of water down to complete spreading, whereas the decrease in θ of glycerol ranges from 9 to 50% of the initial value. For the film modified at the cathode, the total surface energy increases by a factor of 4.7, γp increases by a factor of 6, and γd decreases by a factor of 1.5. For the film treated at the anode, γ and γp increase by factors of 3 and 3.8, respectively, and γd decreases by a factor of 2. These changes in the surface properties of the CPG films correspond in general to
the behavior of the PLLA film under the same condi tions. The main factors responsible for enhancement of hydrophilicity of polymer surfaces as a result of plasma treatment are changes in surface roughness [23] and chemical structure of surface layers owing to the for mation of new functional groups [7, 10]. According to published data [10, 11], modification of polymer films in a lowtemperature air plasma leads to the formation of polar oxygencontaining groups (C–O, –COOH, etc.), resulting in a substantial enhancement of surface hydrophilicity and a noticeable rise in the polar com ponent of the surface energy. To reveal changes in the chemical structure of the surface of CPG films treated on the cathode and anode in dc discharge, an XPS study was performed; its results are given in Table 2. It is seen that the surface layer of the untreated film is enriched in polylactide chains and the O/C ratio of this layer is 0.85, which is close to the O/C value of 0.89 calculated for the chem ical structure of PLLA. Both initial and treated films are characterized by decomposition of the envelope of the C1S peak into three components attributed to the C–C/C–H (284– 285 eV), C–O (286–288 eV), and C=O (288–289 eV) groups (Table 3). However, contrary to expectations, the concentration of oxygencontaining groups decreases after plasma treatment (Fig. 1). It is likely that the enhancement of hydrophilicity of the CPG film by modification is not associated with oxidation processes in surface layers. It seems that during discharge treatment at both the anode and cathode, the degradation process occurs to open the inner film layers, which contain grafted chi tosan chains and gelatin, and results in the appearance of nitrogencontaining groups in the surface layers of the film. The N1S XPS peaks for the films treated on the anode or cathode indicate the formation of N– H/N–O groups during the treatment; moreover, a small amount of nitro groups appears in addition for the sample modified on the cathode (Fig. 2). Based on the XPS data suggesting the degradation of the surface polylactide layers during plasma treat ment, we may assume that the morphological charac teristics of the surface can play a significant role in the enhancement of hydrophilicity of the modified films in this case. Riccardi et al. [12] showed that plasma treatment stronger affects the amorphous region of the polymer, leaving the crystalline phase almost intact. Thus, plasma modification of a sample that includes both amorphous and crystalline structures can lead to an increase in morphological heterogeneity, which is one of the causes of improvement in hydrophilicity of the plasmatreated films. The microphotographs (Fig. 3a) show that the PLLA film has an heterogeneous semicrystalline structure composed of spherulites of 50 to 100 µm in diameter with defect regions between them. The sur HIGH ENERGY CHEMISTRY
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EFFECT OF DIRECTCURRENT DISCHARGE TREATMENT (а)
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(а)
C–O C–C C–H
C=O
N–O N–H
296
288 E, eV
292
(b)
284
280 408
404
C–C C–H
400 E, eV (b)
396
392
N–O N–H
C–O C=O –NO2
296
292
288 284 E, eV
280
276 412
(c)
C–C C–H
408
404 E, eV
400
396
Fig. 2. N1S spectrum of the CPG films treated at (a) the cathode and (b) the anode.
C–O
of local defects of 1 to 3 µm in size, which are presum ably generated during partial degradation of surface layers. The sample modified on the cathode (Fig 3d) is characterized by a noticeably smaller number and size of these defects as compared with the sample treated on the anode (Fig. 3c). According to the published data [7], the treatment of PLLA films in oxygen plasma, depending on the conditions, can cause the appearance of local point defects as in the present work and uniform degradation of the surface layer; this degradation leads to enhancement of surface unifor mity.
C=O
296
292
288 284 E, eV
280
276
Fig. 1. C1S spectrum of (a) the initial CPG film and the films treated at (b) the anode and (c) the cathode.
CONCLUSIONS face of CPG films (Fig. 3b) is characterized by a uni form structure without distinct large crystalline forma tions, which is associated with amorphization pro cesses occurring during solidstate synthesis [24]. The plasma treatment of CPG films leads to the formation HIGH ENERGY CHEMISTRY
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Plasma modification of the chitosan–poly(L,L lactide)–gelatin nanostructured composite material stabilized by the graftcopolymer fraction enhances hydrophilicity and increases the surface energy. According to XPS data, the concentration of polar
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DEMINA et al.
50 µm
(b)
50 µm
(c)
50 µm
50 µm
(d)
Fig. 3. Electron micrographs of the surface of (a) the PLLA film, (b) the initial CPG film, and GPG films treated at (c) the anode and (d) the cathode. The dimensional scale is 50 µm for all micrographs.
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