Surface Microstructure of Chitosan Membranes – AFM Investigations

Polish J. of Environ. Stud. Vol. 15 No. 4A (2006), 84-87Surface Microstructure of ChitosanMembranes – AFM InvestigationsZ. Modrzejewska 1 , J. Stawczyk 1 , K. Matyka 2 , M. Matyka 3 ,I. Mróz 2 , A. Ciszewski 21 Faculty of Process and Environmental Engineering, Technical University of ód,ul. Wólczaska 213/215, 93-005 ód, Poland2 Institute of Experimental Physics, University of Wrocaw, Plac Maxa Borna 9, 50-204 Wrocaw, Poland3 Institute of Theoretical Physics, University of Wrocaw, Plac Maxa Borna 9, 50-204 Wrocaw, PolandAbstractWe present the AFM investigations of chitosan membranes’ surfaces. We observe the surfacemicrostructures and compare the surface areas of chitosan formate and two forms (white and transparent) ofchitosan acetate. The results suggest that the surface of chitosan formate is better developed than the surface ofwhite chitosan aceteate. The transparent chitosan acetate forms the most heterogenous surface.Keywords: chitosan membranes, surface microstructure, AFMIntroductionChitosan (Fig.1a) is a natural polymer revealingunique characteristics that predispose it especially tomedical applications. Chitosan is biocompatible andbiodegradable. It shows athrombogenic and haemostaticproperties. One of chitosan’s most promising features isits excellent ability to form porous structures [1] that areuseful in cell transplantation and tissue regeneration [2].Many possible medical applications of chitosan-derivedmaterials base on adsorption processes that can occur onthe materials’ surfaces. Therefore, the surfacesof chitosan-based membranes, films and multilayers arewidely discussed (e.g. [3-7]). Their microstructures,including roughness etc. can be effectively investigatedusing atomic force microscopy (AFM) and relatedtechniques (e.g. lateral force microscopy LFM [7]).In this paper we use the AFM method to investigatethe surface microstructure of chitosan acetate andchitosan formate membranes. We compare the surfaceareas of the membranes and test usefulness of AFM toinvestigate highly porous materials.Experimental proceduresHydrogel chitosan membranes were produced by thewet phase inversion method. Chitosan, low-viscous,of molecular weight 550 kDa and DD=75% (Fluka, 50494)was solved in acetic acid and formic acid to obtain chitosanacetate and chitosan formate as film forming solutions. Theamount of acetic acid was 0,42 g/g chitosan and the amountof formic acid was 0,3 g/g chitosan . Polymer concentration inthe solution was 5% and 7%. The solution was carried outwith a mixer (shaker, amplitude of oscillations: 8 mm),at temperature 20 o C. After aeration, the solution was pouredonto a flat surface (glass plate) using an applicator witha relevant gap (0.9 mm) that resulted in thin filmsformation. The chitosan membranes were formed byimmersing the thin films, immediately after pouringthe solution onto a flat surface (before the solvent hadevaoporated), into a coagulation bath. The coagulationprocess was carried out in a 10 % aqueous solutionof sodium hydroxide, for 15 minutes. Then the membraneswere washed with water and conditioned in demineralizedwater to obtain pH = 7.0. To preserve their porous structure,

Surface Microstructure of Chitosan… 85oCH 2OHoOHoCH 2OHoOHooCH 2OHoOHoCH 2OHoOHoa)CH 3-CO-NHchitinCH 3-CO-NHnNH 2chitosanNH 2nb)1.4µmHeight /nmHeight /nm1.4µmLateral position/nm200nm200nmc)Lateral position/nmd)Fig. 1. (a) Chitosan [(14)-2 amino-2-deoxy--D-glucane] is a product of chitin [(14)-2 acetamido-2-deoxy--D-glucane]deacetylation. (b) Surface structure of white chitosan acetate, a = 7 m. The standard Height (left) and Deflection (center)images. The profile (right) is taken along the marked line (c) Higher magnification (a =1 m) image shows better bulbousstructures (left–Height, center–Deflection). (d) An example of the dependence between scan size parameter a and Theorigin of the observed effects is discussed in the text. For the same scan sizes, smaller values of correspond to bigger totalsurface areas

86 Modrzejewska Z., et al.the membranes in the form of hydrogel were dried byconvection at low temperatures, in a closed air cycle.Drying of the membranes was carried out at temperatures –4 o C and –12 o C, air circulation was 0.6 ms -1 . For chitosanacetate we observed two forms of the membranes:transparent (created when the thickness of the hydrogelbefore drying was about 0.5 mm) and white (thicknessabout 0.9 mm). The surfaces of the dry membranes wereimaged using the Nanoscope IIIa, Veeco, working in thecontact mode and equipped with an oxide-sharpenedsilicon-nitride probe (NP-S). The length and the springconstant of the applied V-shaped cantilever were 196 mand 0.06 Nm -1 , resp. The measurements were performed inair, at room temperature. The lateral and vertical resolutionswere 10 nm and 1 nm. The AFM images were processedwith the software package WSxM, Nanotec Electronica,Spain [8]. To calculate the total area of an imaged surfacewe approximated the surface by a triangular mesh usingtwo-dimensional linear interpolation of the standard heightmap. Then we calculated a sum of the areas of all thetriangles constituting the mesh. To compare the surfaceareas of chitosan acetate (both forms) and formate, wedefined a dimensionless parameter (as the ratioof the planar area a 2 of an image frame over the total areaof the imaged surface, calculated as described above.Resultsand discussionThe imaged surfaces show that investigated materialsare composed of irregular, bulbous structural elements(Fig.1b,c) of over 100 nm in diameter. The parameterswere calculated separately for chitosan formate and bothforms of chitosan acetate. The results were averaged over30 independent images for a = 20 mand the same numberof independent images for a = 1m. For the imagesof larger area we obtained (mean values av and standarduncertainties – experimental standard deviations of themeans – u()): chitosan formate: av f = 0.9744, u( f ) =0.0008; chitosan acetate (transparent): av act = 0.9771, u( act )= 0.0005; chitosan acetate (white): av acw = 0.9808, u( acw ) =0.0005. The corresponding values for the images of smallerarea are: av f = 0.8762, u( f ) = 0.0071; av act = 0.864, u( act )= 0.013; av acw = 0.9058, u( acw ) = 0.0055. The resultssuggest that the surface of chitosan formate is betterdeveloped than the surface of white chitosan acetate. Thesurface of transparent chitosan acetate should be the mostheterogenous since the corresponding u() obtained for theimages of smaller area is the biggest. For the imagesof larger area, averaging connected with AFM imageformation eliminates the effect of heterogeneity.The presented numerical values are however influencedby several factors. Firstly, the approximation of the surfaceby a triangular mesh eliminates structural details andreduces the calculated total surface area. The effect is strongerfor the images of larger area. Secondly, the qualityof surface imaging depends on the scanning velocity. Forrough and extended surfaces, the optimal scanning frequencyis about 1 Hz. If the same frequency is applied for scanningof the regions of different areas, the corresponding tipvelocity changes. We tested the influence of the scanningrate on the surface corrugation registered on the AFMpatterns and we can conclude that the image of the samearea (a = 10 m) obtained using faster scanning looksflatter than those obtained using slower one (the structuraldetails of the surfaces are eliminated if the scanning is toofast). Both factors mentioned above explain the influenceof the dimension of the scanned area on . The typicalrelation we observed is presented in Fig. 1 d. Themeasurements were done for the same scanning frequency,for a = 1,2,…,20 m. Significant deviations from thepresented plots are possible for the smallest scans,especially for transparent chitosan acetate.Another effect that is usually observed for surfacesof materials composed of spherical/bulbous elements isrelated to the shape of the used cantilever. In this case theimaged surfaces are “flatten” and their total areas of thesurfaces are smaller. Finally, we have some dependence onsoftness of the material that may be deformed by the tipduring the measurement. The reasons listed above indicatethat numerical values of can be treated as approximateones. The observed tendencies are however promising andmay give us valuable information about structuralproperties of chitosan membranes.ConclusionsBasing on the performed AFM investigations wesuggest that for three types of chitosan membranes: chitosanacetate (white and transparent) and chitosan formate, thesurface area of chitosan formate is bigger than the surfacearea of white chitosan acetate. The transparent formof chitosan acetate is characterized by the most heterogenoussurface.References1. Berger J., Reist M., Mayer J.M., Felt O., Gurny R.:Structure and interactions in chitosan hydrogels formedby complexation or aggregation for biomedicalapplications. Eur. J. Pharm. and Biopharm. 57 (1), 35,2004.2. Lu G., Zhu L., Kong L., Zhang L., Gong Y., Zhao N.,Zhang X.: Porous chitosan microcarriers for large scalecultivation of cells for tissue engineering: Fabricationand evaluation. Tsinghua Sci. Technol. 11 (4), 427,2006.3. Nosal W.H., Thompson D.W., Yan L., Sarkar S.,Subramanian A., Woollam J.A.: UV–vis–infraredoptical and AFM study of spin-cast chitosan films.Colloid Surfaces B 43 (3-4), 131, 2005.

Surface Microstructure of Chitosan… 874. Nosal W.H., Thompson D.W., Yan L., Sarkar S.,Subramanian A., Woollam J.A.: Infrared optical propertiesand AFM of spin-cast chitosan films chemicallymodified with 1,2 Epoxy-3-phenoxy-propane. ColloidSurfaces B 46 (1), 26, 2005.5. Feng Y., Han Z., Peng J., Lu J., Xue B., Li L., Ma H.,Wang E.: Fabrication and characterization of multilayerfilms based on Keggin-type polyoxometalate andchitosan, Mater. Lett. 60 (13-14), 1588, 2006.6. Aimoli C.G., Torres M.A. Beppu M.M.: Investigationsinto the early stages of “in vitro” calcification onchitosan films. Mater. Sci. Eng. C 26 (1), 78, 2006.7. Fu J., Ji J., Yuan W., Shen J.: Construction of antiadhesiveand antibacterial multilayer films via layerby-layerassembly of heparin and chitosan. Biomaterials26 (33), 6684, 2005.8. WSxM free software downloadable athttp://www.nanotec.es.