Modification of Polylactide Surfaces with Lactide-Ethylene Oxide

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Biomacromolecules 2010, 11, 68–75

Modification of Polylactide Surfaces with Lactide-Ethylene Oxide Functional Block Copolymers: Accessibility of Functional Groups Elisˇka Trˇesohlava´,* Sˇteˇpań Popelka, Ludka Machova´, and Frantisˇek Rypaćˇek Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky sq. 2, Prague 6, 162 06, Czech Republic Received August 10, 2009; Revised Manuscript Received November 13, 2009

Feasibility of using amphiphilic block copolymers composed of polylactide (PLA) and poly(ethylene oxide) (PEO) blocks for biomimetic surface modification of polylactide-based biomaterials for tissue engineering was investigated. PEO-b-PLA copolymers were deposited on the PLA surface from a solution in PEO-selective solvent. Copolymers with a neutral ω-methoxy end group of the PEO block (mPEO-b-PLA) were used to provide hydrophilic surface of PLLA, which exhibited suppressed nonspecific protein adsorption. Their analogues, containing biotin group at the end of PEO block (bPEO-b-PLA), were used as a model of functional copolymers, carrying a biomimetic group, for example, a cell-adhesion fibronectine-derived peptide sequence. The surface topography of functional groups on the modified surface and their accessibility for interaction with a protein receptor was investigated, taking advantage of specific biotin-avidin interaction, on surfaces modified with a combination of mPEO-b-PLA and bPEO-b-PLA copolymers. The accessibility of model biotin groups for interaction with their protein counterpart was proven through visualization of avidin or avidin-labeled nanospheres with atomic force microscopy.

Introduction Biomaterials for guided tissue regeneration and tissue engineering are required to have well-defined surfaces that would provide for controlled cell-biomaterial interactions. To this end, surface-bound bioactive structures, mostly extracellular matrix (ECM) components or their mimics, for example, peptide sequences derived from ECM proteins such as fibronectin or laminin, are needed. The biomimetic structures on the surface facilitate functional cell adhesion, migration, growth, or differentiation.1-5 The effect on cell response of the presence and density of bioactive ligands on biomaterial surfaces is under frequent investigation.2,3,5-16 In addition, also the microenvironment of immobilized functionalities was proved to be an important factor in effective biomaterial-cell receptor interactions.14,17 However, to take advantage of the specifically attached biomimetic groups, the tested surface must be first resistant to nonspecific protein adsorption, otherwise, the presentation of biomimetic motifs would be masked by adsorbed layer of proteins that occurs at the first instant after the biomaterial is brought into contact with a protein-containing biological fluid, such as body environment or tissue culture medium. The present work is focused on the surface modification of polyesters biomaterials. Among the biodegradable polymers in the tissue engineering field, aliphatic polyesters, such as polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and their copolymers are the most often studied.17,18 They are nontoxic and degradable within an adjustable time, and for tissue scaffolds, they can be easily processed to fibres, films, or 3D porous structures. They adsorb proteins very well, due to their hydrophobic character. To control protein adsorption, surfaces need to be modified with tethered flexible hydrophilic polymers. Tethering hydrophilic poly(ethylene oxide), PEO, is proven to be effective in the creation of surfaces * To whom correspondence should be addressed. Fax: +420-296809410. E-mail: [email protected].

resistant to protein adsorption, and in this respect, it has been already used for various biomedical applications.5,17-19 Various techniques for immobilization of PEO have been reported, for example, surface grafting, physical entrapment, and deposition of PEO-block copolymers, and the effect of PEO on the resistance of surface to nonspecific protein adsorption was investigated.2,8-13,19-28 In case of polyesters, because their low content of suitable reactive groups, physical techniques for immobilization of PEO on their surfaces are more suitable. In addition to the creation of nonfouling surfaces, some of the tethered PEO chains can be also used to carry functional groups, for example, bioactive components bound to the chain end group.2,7,9,10,18,19,26 Numerous strategies have been developed and are under further investigation for coupling of bioactive compounds to polymers for drug delivery and tissue engineering. Besides extracellular matrix proteins and their derivatives, other structures such as carbohydrates, nucleotides, viruses, or liposomes have been used.6,17,29-38 Preparation of conjugates of (co)polymers with bioactive molecules via different controlled polymerization techniques or by using various coupling reactions have been thoroughly reviewed by Lutz and Borner.39 For tissue-engineering biomaterials, diverse techniques for surface pattern fabrication in terms of roughness40-42 as well as immobilization of signaling molecules were developed, including etching, various lithographic, irradiation, or grafting techniques.17,43-48 When covalent immobilization of protein molecules on the chemically inert polymeric biomaterials is performed, reactive groups serving as coupling sites such as hydroxyl or amino groups must be introduced, often by various irradiation techniques in the presence of some chemical agent (e.g., H 2O 2), followed by “grafting to” or “grafting from” procedures.15,17,27,43-45,49 The hydrolysis and aminolysis was also used for production of reactive groups on the polyester surface,44,50,51 however, such procedures may result in

10.1021/bm900889b  2010 American Chemical Society Published on Web 12/02/2009

Modification of Polylactide Surfaces

polyester molecular weight changes. Other techniques based on physisorption, mainly on entrapment of biomolecules or their conjugates with hydrophilic polymers (e.g., PEO) to the solid polymer surface as well as self-assembly of a thiolcontaining compound on the gold surface are also frequently used.15,23,27,28,44,46,52 Though these state-of-the-art procedures can be successfully applied to the well-defined geometry of planar surfaces (gold, silica, glass), they can hardly be used for the development of biomimetic surface patterns on real biomaterial surfaces, in a wide range of densities and arrays of peptide motif clusters in a nanometer-scale on complex 3D shapes. In our previous study,53 we investigated the efficiency of polylactide (PLA) surface modification using a surface deposition of PLA-b-PEO amphiphilic block copolymers. The PLAb-PEO copolymers were deposited from aqueous media or polar selective solvents in which they can form micelles or, generally, colloidal self-assemblies, typically with a core-shell structure, where the core is composed of hydrophobic (PLA) blocks, whereas the shell is constituted from hydrophilic (PEO) blocks. Despite that the self-assemblies in selective solvents are formed under conditions of dynamic equilibrium, it has been proven in our earlier studies that the appropriate selection of molecular parameters of PLA-b-PEO block copolymers may lead to nanocolloids that are relatively kinetically stable.54 Taking advantage of good miscibility of PLA chains in both the hydrophobic block of the diblock copolymer and that of the modified solid, the PLA-b-PEO copolymer layer deposited on PLA surface is stabilized by anchoring interactions between the surface and the chains in the particle core.53-56 The formation of the copolymer layer, the surface density of thus obtained tethered PEO chains, and its efficiency in preventing the nonspecific protein adsorption compared to unmodified PLA surfaces was investigated by Popelka et al.53 and related to the molecular parameters of diblock copolymers, such as molecular weight and block ratios. In the present study we use the deposition of PLA-b-PEO block copolymers not only to suppress nonspecific protein adsorption on PLA surfaces, but also to introduce selected bioactive structures to these neutral surfaces. We studied whether, after deposition of copolymers on the surface, the functional groups bound to the end of PEO chain of the block copolymer would remain accessible for a molecular interaction with a specific protein structures such as cell trans-membrane receptors, despite that a general protein adsorption was suppressed. As a model system for these studies we use PLA surfaces modified with mixtures of copolymers composed of a neutral PLA-b-PEO and its analogue with biotin at the end of PEO block. In this model, biotin serves as a surrogate of a bioactive peptide group, for example, sequence of amino acids derived from fibronectine such as arginine-glycine-aspartic acidserine (conventionally labeled as RGDS). The accessibility of such a functional group at the end of the PEO chain of the deposited PLA-b-PEO copolymer layer is investigated by following the interaction of biotin, as a reporting group, with avidin. Avidin, a 66-kDa glycoprotein found in egg white, and its derivatives streptavidin and neutravidin have a strong affinity for biotin, forming one of the strongest protein-ligand complexes with a dissociation constant of 10-15 M. The avidin-biotin system has been widely used as a model to study macromolecule-ligand interactions or biomolecules labeling in immunohistochemistry and molecular biology.57-59 Taking advantage of strong affinity of avidin to biotin, the accessibility of biotin

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Figure 1. Synthesis of biotinylated poly(ethylene oxide) (Biotin-PEOOH).

as a functional group can be followed by detecting of the much larger avidin molecules bound to biotin moieties using atomic force microscopy (AFM).7,60 Feasibility of using AFM and avidin-biotin detection system for investigation of surface topography of biomimetic groups on the surfaces modified with adsorbed amphiphilic block copolymers is evaluated.

Experimental Section Materials. Dimethyldichlorosilane (Fluka), D-biotin p-nitrophenyl ester (Sigma), potassium carbonate, R-amino-ω-hydroxy-polyoxyethylene (Mw ) 3400; Shearwater Polymers), R-aminopropyl-ω-hydroxypolyoxyethylene (Mw ) 10300; NOF Corporation), avidin from egg white (Sigma), FluoSpheres-NeutrAvidin labeled microspheres (diameter 40 nm, 2.28 × 1014 particles per mL, Invitrogen), and Tween 20 (Fluka) were used as purchased. Milli-Q deionized water was used for all aqueous solutions and procedures. Tin(II) 2-ethylhexanoate (Sn(Oct)2; Aldrich) was distilled under vacuum and stored under inert gas at -18 °C. Triethylene glycol monomethyl ether (Fluka) was two times distilled over LiAlH4 under reduced pressure (80 °C, 0.3 kPa). Ethylene oxide (Fluka) was dried for 3 days over CaH2 in a storage glass ampule equipped with a brass vent. L-Lactide and DL-lactide (Aldrich) were used freshly recrystallized from a mixture of ethyl acetate and toluene. Poly(L-lactide) (PLLA) was synthesized by ring-opening polymerization of L-lactide in melt, with Sn(Oct)2 as a catalyst. The polymerization was carried out at 110 °C for 60 h. The resulting polymer was dissolved in dichloromethane and isolated by precipitation in methanol and filtration. The crude product was further purified by reprecipitation twice from solution in dichloromethane into methanol. R-Hydroxy-ω-methoxy-poly(ethylene oxide) (MeO-PEO-OH) with different molecular weight was synthesized by anionic polymerization of ethylene oxide as described elsewhere.54 Biotin-PEO-OH: R-Amino-ω-hydroxy-polyoxyethylene (I, Figure 1) (450 mg, 4.4 × 10-5 mol NH2) was dissolved in dimethylformamide (DMF) (6 mL) and mixed with a solution of d-biotin p-nitrophenyl ester (II; 23.9 mg, 6.5 × 10-5 mol) in DMF (6 mL) and triethylamine (4.86 µL, 3.5 × 10-5 mol) was added. The reaction solution was stirred under laboratory temperature for 72 h, DMF was evaporated in a vacuum evaporator and the “oily residue” was dissolved in a potassium carbonate solution (0.5 mol · L-1). The pH of solution was adjusted to 12 and the solution was desalted and washed with water by membrane ultrafiltration (YM3, Amicon Corporation). The product (III) was obtained by lyophilization and stored under inert gas in a refrigerator. PDLLA-PEO-biotin, PDLLA-PEO-OMe (Figure 2): Diblock copolymers composed of poly(DL-lactide) and poly(ethylene oxide) blocks with either methoxy- or biotinyl-group at the end of PEO block and with different PDLLA and PEO block lengths were prepared by controlled ring-opening polymerization of DL-lactide (I) in toluene using heterobifunctional MeO-PEO-OH or Biotin-PEO-OH, respectively, as macroinitiators and Sn(Oct)2 as a catalyst. The catalyst-to-macroinitiator

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Trˇesohlava´ et al. Table 1. Composition of Spin-Cast Copolymer Solutions and Corresponding Codes of Sample Surfacesa surface code mE5L5 mE5L5-bE4L5 mE15L5 mE15L5-bE10L5 mE15L5-bE10L5-low

spin-cast solution composition neutral copolymer mE5L5 90 wt % of neutral mE5L5 + 10 wt % of functionalized bE4L5 neutral copolymer mE15L5 90 wt % of neutral mE15L5 + 10 wt % of functionalized bE10L5 99.5 wt % of neutral mE15L5 + 0.5 wt % of functionalized bE10L5

a The final copolymer concentration in dioxane/methanol 1/9 vol % was 5 mg/mL.

Figure 2. Synthesis of functional poly(DL-lactide)-block-poly(ethylene oxide) copolymers.

molar ratio was 3.4:1. The required amount of DL-lactide monomer was calculated with respect to the molecular weight of macroinitiator (MPEO) and desired length of polylactide block (MPLA ) 5000 g/mol) and used in 20% surplus to the calculated value. The polymerization was carried out at 90 °C for 18 h, while stirring under a nitrogen atmosphere in ampules equipped with a condenser and a three-way stopcock through which the apparatus was connected to a vacuum/ inert gas manifold. The reaction product was poured into diethyl ether and the precipitated polymer was isolated by filtration. The dried products (III) were stored under inert gas at low temperature. Methods. Sample Surface Preparation. As a supporting surface, representing the surface of polyester biomaterial, a poly-L-lactide (PLLA) film cast on a glass slide was used. To facilitate a firm adhesion of the PLLA film, the glass surface was first hydrophobized by reaction with dimethyldichlorosilane. The functional PDLLA-b-PEO copolymer layer was then deposited on top of the PLLA film, using spin-casting from solution mixtures containing different ratios of neutral/functionalized copolymers. The preparation steps in detail were as follows. Silanization of glass slides: Glass slides (circular, diameter 12 mm) were washed in petrolether (2 × 15 min) and methanol (2 × 15 min), both under sonication in ultrasonic bath, and dried at 100 °C for 20 min. The final cleaning was done in Harrick Plasma Cleaner, using air plasma for 5 min. Freshly cleaned slides were immersed in Milli-Q water to hydrate the surface and then put in silanization solution consisting of 50 µL of dimethyldichlorosilane per 100 mL of toluene. The reaction with silane was carried out for 1 h at room temperature while shaking. The silanized slides were washed two times with toluene, once with methanol, dried for 20 min at 100 °C, and stored under vacuum. Spin-casting of PLLA film: The PLLA films on silanized glass substrates were spin-cast from polylactide solution in chloroform (5 mg/mL) using PWM32-PS-R790 spinner (Headway Research, Inc.). The spin-cast films were annealed at 70 °C for 1 h under vacuum. Spin-casting of copolymers: The PLA-b-PEO copolymer films on PLLA were prepared by spin-casting of copolymer solutions, containing neutral and functionalized copolymers in selected ratios, as listed in Table 1, in a methanol/dioxane (9:1 v/v) mixed solvent, with copolymer concentration of 5 mg/mL. A total of 60 µL of a given solution was applied on each slide and spin-cast (2500 rpm, 30s). Spin-cast copolymer films were annealed in Milli-Q water for 1 h at 60 °C and quenched by immersing into ice-cold Milli-Q water afterward. The coated slides were dried at room temperature under vacuum (3 Pa) overnight and stored under vacuum. All the film preparation procedures were carried out in a dust-free laminar-flow box.

Surface Reaction with AVidin. Spin-cast polymer films (including PLLA film) were first immersed in a 0.01 mol · L-1 phosphate buffered saline solution (PBS), pH ∼ 7.4, for 5 min to equilibrate and then incubated in an avidin solution (0.15 mg/mL) in PBS for 30 min at 25 °C. The avidin-treated surfaces were washed two times with PBS solution and three times with Milli-Q water. Washed samples were dried overnight at room temperature under vacuum and stored in desiccator in darkness until their characterization. Surface Reaction with NeutrAVidin-Labeled Microspheres. The reaction with avidin-labeled microspheres was performed on surfaces prepared from copolymers with longer PEO chain and with lower surface content of functional/biotin group (mE15L5-bE10L5-low). The original dispersion of NeutrAvidin-labeled microspheres was sonicated for 30 min to break up any potential aggregates. Then 20 µL of the dispersion was diluted with 19.98 mL of PBS containing 1% v/v of Tween 20 and sonicated to secure a homogeneous dispersion of particles. The reaction of microspheres with surfaces was carried out in the same way as the above-described reaction with avidin alone (30 min, 25 °C). The surfaces were then washed, dried, and stored as mentioned above for pure avidin. Nuclear Magnetic Resonance Spectroscopy (NMR). 1H NMR spectra of copolymers in CDCl3 were acquired using high-resolution NMR spectrometer Bruker DPX300 in CDCl3 at 330 K. Size Exclusion Chromatography (SEC). SEC was performed on Waters HPLC-SEC modular system, using combined PLgel 103 Å, 10 µm, and PLgel Mixed-C (2 × 7.5 × 600 mm) columns in tetrahydrofuran (THF), with Waters 410 RI detector. The columns were calibrated with PEO standards (Chrompack) for PEO measurements. For PLA, the columns were calibrated using polystyrene standards and the calibration was recalculated for PLA using Mark-Houwink coefficients for polystyrene and PLA61 and a universal calibration concept.62 Values of number average and weight average molecular weight, Mn and Mw, were determined. Contact Angle Measurement (CAM). The static water/air contact angle was determined by sessile drop method on the Contact Angle Measuring System OCA_20 (Dataphysics, Germany) using OCA software SCA 20. A 2 µL drop of deionized water Milli-Q was applied to the surface and contact angle measurements were performed on opposite edges of the drop using Laplace-Young fitting as a calculation method. The measurements were repeated at least with four drops on different places of each substrate, and the minimum number of substrates prepared for characterization of each type of layer was five. The water/air contact angle was calculated as an average from all measurements and the standard deviation was determined (based on at least 20 independent measurements). Atomic Force Microscopy (AFM). All images were acquired with Multimode Atomic Force Microscope Nanoscope IIIa (Digital Instruments) as topological scans in tapping mode in air, using tapping mode etched silicone probes OTESPA (Veeco Instruments) with cantilever resonance frequencies about 350 kHz. The 512 × 512 pixel images were scanned at a rate of 1 Hz. The images were obtained both before and after incubation of samples with avidin labeling solutions.



Table 2. Molecular Characteristics of Diblock Copolymers copolymer Mn (PEO)a Mn (PDLLA)b Mn (total) Mn/Mwa fc (%) mE5L5 mE15L5 bE4L5 bE10L5

5600 15100 3400 10300

4240 5800 4800 4500

9840 20900 8200 14800

1.12 1.05 1.20 1.10

82 98

a Determined by SEC. b Determined by 1H NMR. c The percentage of PEO block end groups occupied with biotin as determined by 1H NMR.

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Table 3. Water/Air Contact Angle Θstat with Standard Deviation of the Original and the Modified PLLA Surfaces surface

Θstat (deg)

silanized glass slide PLLA film mE5L5 mE5L5-bE4L5 mE15L5 mE15L5-bE10L5 mE15L5-bE10L5-low

101 ( 0.8 78 ( 0.5 69 ( 3.7 73 ( 2.4 53 ( 2.6 56 ( 2.6 50 ( 3.6

Results and Discussion Polymer Synthesis and Characterization. High-molecularweight homopolymer PLLA, and A-B diblock copolymers composed of poly(DL-lactide) (PDLLA) and poly(ethylene oxide) (PEO) blocks with biotin at the end of PEO block and with different block lengths were synthesized by ring-opening polymerization and characterized by 1H NMR spectroscopy and size exclusion chromatography (SEC; Table 2). Copolymers of poly(DL-lactide) and poly(ethylene oxide) with methoxy group at the end of PEO block, with matching PEO and PDLLA block lengths, were used as a neutral environment of functional copolymers and were prepared and characterized under similar condition (Table 2). The copolymer composition, that is, the ratio of both blocks (PEO, PDLLA) have been calculated from the ratio of integrated peaks of methylene protons of PEO unit (3.54 ppm) and that of PLA-methine proton (5.2 ppm). In the case of functionalized copolymers, biotin was coupled to the PEO via reaction of D-biotin p-nitrophenyl ester with amino group at the end of PEO chain prior to the polymerization of lactide (Figure 1). The biotin group was identified at 4.2 and 4.3 ppm in 1H NMR as signals of two methine protons from the cyclic biotin structure. The biotin attachment was confirmed by the presence of triplet at 2.2 ppm spectrum that is attributed to the methylene protons of the biotin chain in R position to the amide bond (Figure 1) and the degree of copolymer functionalization was calculated from this integrated peak. Surface Preparation and Modification. The study was performed as 2D model on glass slides. The created (co)polymer layers were characterized after each step of modification by contact angle measurements and atomic force microscopy (AFM). The glass surface was first hydrophobized by reaction with dimethyldichlorosilane to provide good adhesion of cast polylactide film. The hydrophobic character of underlying silane modification was confirmed by contact angle measurements (see below) and the AFM observations confirmed smooth surface

in a nanometer scale (data not shown). Subsequent coating with polylactide, by casting from PLLA solution, provided rather smooth PLLA film with thickness of about 30 nm, as it is depicted in Figure 3a. In this 2D model, the PLLA film represents the surface of a hypothetical polylactide scaffold. PDLLA-b-PEO copolymers with different block lengths were then spin-cast on the top of PLLA film, either as a solution containing only neutral copolymer, or a mixture of neutral and the functional copolymer containing biotin at the end of PEO block, with matching PEO and PLA block lengths. The integrity of acquired (co)polymer films was confirmed by light microscope observations prior to AFM measurements. The surfaces created from a neutral copolymer only were expected to withstand nonspecific protein adsorption, including avidin, and in the following study with biotin containing surfaces were used as a reference. The accessibility of biotin functional groups on surfaces containing both neutral and biotin-functionalized copolymer was studied using avidin or NeutrAvidinlabeled nanospheres, representing here an approaching cell receptor, taking advantage of a well-known specific interaction between avidin and biotin. The accessibility of biotin groups at the end of the PEO block was proved by detecting much larger avidin bound to biotin by AFM as described below. Surface PropertiessWettability. The changes in surface energy, that is, in surface wettability, were used as a fast and reproducible tool for monitoring the gross effect of sample surface modification. All surfaces were characterized by static contact angle measurement, using at least four drops on each substrate. The static “sessile drop” method was selected with respect to the sample size. The average contact angle values for studied surfaces are compared in Table 3. The average value for PLLA corresponds well with values for PLLA given in other studies.63-65 As it can be expected, coating of PLLA with PDLLA-b-PEO copolymers resulted in more hydrophilic surfaces, that is, exhibiting lower contact

Figure 3. Tapping mode AFM images, 1 × 1 µm, Z-scale 100 nm, of PLLA surface taken (a) before and (b) after incubation of samples with the avidin solution.

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Figure 4. Tapping mode AFM images, 1 × 1 µm, Z-scale 100 nm, of neutral copolymers mE5L5 (a) and mE15L5 (b) deposited on the supporting PLLA film.

angle values. The change in wettability with respect to original PLLA is lower for the copolymers with a shorter PEO block. No significant difference in wettability was found between surfaces modified with copolymers mE15L5 and mE10L5, with Mn of PEO blocks 15100 and 10300, respectively. The addition of the biotinylated variant (bE10L5) to the neutral copolymer did not change significantly the surface wettability, indicating that the presence of a small fraction (10 or 0.5%) of biotin functional groups has no significant effect on the gross surface properties. Surface Topography and Functional Group Detection. The surface topography was followed by atomic force microscopy (AFM) in tapping mode under ambient condition and it was related to the surface composition and to the molecular parameters of copolymers. Topography images were taken after each step of modification, thus including unmodified PLLA surface as well as PLLA modified with copolymers, both before and after the incubation with avidin solution or with NeutrAvidin-labeled nanospheres. Figure 3a shows a 1 × 1 µm tapping mode scan of a typical PLLA surface. It shows that relatively smooth and stable polylactide films were prepared. Figure 3b shows the PLLA surface after incubation with the avidin solution. The protein (avidin) adsorption on the PLLA film is clearly evident as a deposited mass of protein aggregates on the surface, compared to relatively smooth surface of PLLA seen in Figure 3a. The morphology of surfaces created by spin-casting the neutral diblock PDLLA-b-PEO copolymers with different poly(ethylene oxide) block lengths on top of the PLLA film is presented in Figure 4. The deposited copolymer film resulted in surfaces with higher roughness. From a comparison of Figure 4a and b, it is clearly seen that the surface morphology in detail depends on molecular parameters of the deposited copolymer. The copolymer with shorter PEO block, for example, mE5L5, form smaller, rather unorganized spherically shaped deposits while the copolymer with longer PEO block, for example, mE15E5, tends to form a more structured surface morphology with a distinct lamellar pattern. This difference can be explained by the tendency of longer PEO chains to crystallize and thus to a more complete phase separation of polylactide and poly(ethylene oxide) phases. The previous study66 of thermal properties of PEO/PLA block copolymers indicates that the range of microphase compatibility depends on the ratio of PEO/PLA in the copolymer, and it may play a significant role in the formation of the structured surface. Based on these data we can assume that in the mE5L5 surface layer, both copolymer blocks can be mixed together with no or

incomplete phase separation. On the other hand, in the case of the copolymer with longer PEO block length (mE15L5), the PEO block and PDLLA block are likely to separate more completely. Because of the amphiphilic character of diblock copolymers and taking into account obtained water contact angle values, we can assume that the separated polylactide block of mE15L5, thus, the PLA core of its aggregates, preferentially tend to adsorb to the supporting PLLA surface, and the PEO block can crystallize, creating the dominant hydrophilic surface character. The formation of PEO domains in copolymer film cast on the water surface was confirmed in a previous study66 using transmission electron microscopy together with osmium tetraoxide for contrasting the PEO phase. The morphology result indicates that the PLA-b-PEO copolymers undergo a microphase separation that is dependent on the PLA/PEO ratio in copolymer composition mentioned above, on the stereoregularity of PLA block, and on the method of preparation. Based on the known nonfouling character of PEO,18,19 a previous observation by Popelka,53,54 and our results obtained by atomic force microscopy presented in Figure 5, we can conclude that the pronounced nonspecific protein adsorption to pure PLLA (shown in Figure 3b) can be significantly suppressed by coating PLLA with PLA-b-PEO copolymers, in particular, copolymers with longer PEO blocks. Figure 5 shows a copolymer surface under ambient condition made by spin-casting of mE15L5 (Figure 5a), and in Figure 5c, the surface made from solution in which a 10 wt % fraction of neutral copolymer mE15L5 was substituted with the copolymer containing biotin at the end of PEO chain (bE10L5). Acquired 1 × 1 µm scans of original copolymer surfaces (Figure 5a,c) were compared with copolymer surface topography pictures taken after their incubation with avidin solution (Figure 5b,d). As it was mentioned above, the biotin group serves as a model surrogate representing an adhesion peptide sequence. Taking advantage of strong affinity of avidin to biotin, the accessibility of biotin as a functional group was followed by detecting of much larger avidin molecules bound to biotin. The original neutral surface mE15L5 (Figure 5a) as well as the surface functionalized with biotin mE15L5-bE10L5 (Figure 5c) exhibited a lamellar structure in dry state. The neutral surface mE15L5 retained its lamellar character even after incubation with avidin solution (Figure 5b). Slightly different lamellae organization can be attributed to the history of the sample, for example, the stay of surface in aqueous medium during incubation with



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Figure 5. AFM tapping mode topography scans 1 × 1 µm, Z-scale 100 nm, taken under ambient condition of mE15-L5 copolymer surface (a,b) and functionalized surface m+bE10-L5 (c,d). In the micrographs (a) and (c) are copolymer surfaces before incubation and (b) and (d) show surfaces after the incubation with the avidin solution.

protein solution and following rinsing and surface redrying, which may result in PEO chain reorganization. This finding, together with results of a previous study53 and with presented surface wettability characteristics, indicates that the PEO phase is exposed on the surface when the surface is immersed in water; during incubation with protein solution, the PEO chains are swollen, expanded, and can play its role in reducing protein adsorption compared to unmodified PLLA surface in Figure 3. In contrast to a neutral mE15L5 surface without biotin groups, on the biotinylated surface of mE15L5-bE10L5 (Figure 5d) the distinct lamellar character of the surface disappeared after the incubation with avidin, with fine lamellar structure of the copolymer being covered with the bound protein. Compared with the effect of avidin on the neutral surface (Figure 5b), it is evident that the avidin binding to biotinylated copolymer is not caused by a nonspecific adsorption but rather due to specific binding to the present biotin groups. This indicates that the biotin groups during incubation in aqueous solution are exposed on the surface and are well accessible for the interaction with avidin molecules. The availability of surface functional groups for the interaction with protein as a part larger structure was confirmed by experiment using analogous strategy with avidin bound to nanospheres (NeutrAvidin labeled microspheres, diameter of about 0.04 µm, Molecular Probes). The surface with lower content of biotinylated copolymer was used to investigate the feasibility of localizing the individual functional groups. Figure

6a shows the appearance of sporadic spheres adsorbed on the smooth original PLLA surface together with relevant surface profile, confirming the particle size given by producer. The presence of functional biotin groups and their accessibility on the surface was proved by incubation of mE15L5bE10L5-low surface with NeutrAvidin-labeled nanospheres, which were subsequently visualized on the functional surface (Figure 6c), thus providing an evidence that functional biomimetic groups, when attached to amphiphilic PLA-b-PEO copolymers, are accessible for the interaction with their protein counterparts, possibly a receptor on cell surfaces. The AFM technique with labeled nanospheres as markers can be used for monitoring surface topography and distribution pattern of biomimetic structures. These studies are in progress and will be subject of next communication.

Conclusions Polylactide surface was modified by deposition of amphiphilic diblock copolymers of PLA and PEO from PEO-selective solvent. The effect of deposited copolymer layer on gross physical properties of the surface was characterized by contact angle measurement and atomic force microscopy. The morphology and wettability of surfaces modified by amphiphilic block copolymers depend on the molecular parameters of used copolymers. The surfaces modified with copolymers composed of PEO with MW 10 and 15 KDa were hydrophilic and exhibited markedly increased wettability and

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Figure 6. Tapping mode AFM images and their representative profiles of (a) microspheres on PLLA (scan size 1 × 1 µm, Z-scale 50 nm) and (b,c) of copolymer surface mE15L5-5bE10L5-low (scan size 5 × 5 µm, Z-scale 100 nm). The micrograph (b) was taken before, and the picture (c) after the incubation with marker: white lines, profile paths; arrows, NeutrAvidin labeled microspheres bound to the biotinylated copolymer.

decreased nonspecific adsorption of proteins with avidin as a protein example. The deposited copolymer layer was stable enough to afford incubation in aqueous buffers and extensive washing for specific protein binding studies. When AFM and the biotin-avidin strategy were used in which biotin served as a reporting group mimicking an adhesion peptide at the end of PEO block, the presence of the biotin groups on the modified surface was clearly manifested by enhanced binding of avidin or avidin-labeled nanospheres. Selective binding of avidin to biotinylated copolymers provides evidence that functional biomimetic groups on the surfaces modified by amphiphilic PLA-b-PEO copolymers are exposed on the surface and, hence, can be accessible for the interaction with their protein counterparts, possibly receptors on cell surfaces.

When larger markers are used, such as avidin-labeled nanospheres, AFM and potentially other microscopic techniques are promising techniques for monitoring the surface topography of individual functional groups. Acknowledgment. The support by Academy of Sciences of the Czech Republic (Grant No.: 1QS500110564) and EXPERTISSUES (6th FP NoE No.: NMP3-500283-2) is acknowledged.

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