Communication

DOI: 10.1002/marc.200600223

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Summary: Stimuli-responsive glycopolymer brushes composed of N-acryloyl glucosamine (AGA) and N-isopropylacrylamide (NIPAAm) were prepared using RAFT polymerization. The RAFT agent was immobilized on the surface of a treated silicon waver via covalent attachment using the Z-group. PAGA and PNIPAAm brushes showed a linear increase in brush thickness with the consumption of monomer in solution. The polymers generated in solution conrm the living behavior with the molecular weight increasing linearly with monomer conversion while the molecular weight distribution remains narrow. Additionally, the ability of PAGA brushes to grow further in the presence of NIPAAm reveals the presence of an active RAFTend group indicative of a living system. PAGA and PNIPAAm homopolymer brushes up to 30 nm were grown using this technique. PAGA brushes were utilized for further chain extension to generate stimuli-responsive brushes with block structures of PAGA and PNIPAAm. The PAGA-blockPNIPAAm brushes were found to grow in size with the consumption of NIPAAm. Contact angle measurements conrm the suggested mechanism showing that the second monomer is incorporated between the rst layer and the silicon surface as expected using the Z-group approach.

Structure of the stimuli-responsive glycopolymer brushes.

Temperature-Responsive Glycopolymer Brushes Synthesized via RAFT Polymerization Using the Z-group Approacha
Martina H. Stenzel,*1 Ling Zhang,1 Wilhelm T. S. Huck2
1

Centre for Advanced Macromolecular Design (CAMD), School of Engineering and Industrial Chemistry, The University of New South Wales, Sydney NSW 2052, Australia E-mail: M. Stenzel@unsw.edu.au 2 Melville Laboratory for Polymer Synthesis, Department of Chemistry, Cambridge University, Cambridge CB2 1EW, UK

Received: March 29, 2006; Accepted: May 2, 2006; DOI: 10.1002/marc.200600223 Keywords: biomaterials; glycopolymer; graft copolymers; reversible addition fragmentation chain transfer (RAFT); stimulisensitive polymers

Introduction
The modication of surfaces is a versatile way to alter the interaction of a material with its environment without changing the bulk properties. This can be important for materials for biomedical applications where a high mechanical stability or stability against degradation is
a

: Supporting information for this article is available at the bottom of the articles abstract page, which can be accessed from the journals homepage at http://www.mrc-journal.de, or from the author.

prerequisite for the bulk material while the surface requires biocompatible properties to inuence the adsorption of proteins. The surface chemistry plays a major role in the interaction of the material with its environment. Proteins have a tendency to adsorb onto surfaces either in a specic way using receptor-ligand interactions or via non-specic binding, thus, determining if a material such as an implant is biocompatible or not.[1] Surfaces can be modied using a range of techniques such as the adsorption of LangmuirBlodgett layers, the layer-by-layer technique or the grafting from or onto polymerization. Especially the grafting from or

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onto technique results in stable polymer brushes with a high stability against erosion.[2,3] In the grafting from approach, an initiator bound to the surface enables an easy control on the thickness and density of the polymer brush using a range of techniques such as living ring opening polymerization, anionic polymerization or controlled radical polymerization techniques such as NMP, ATRP, and reversible addition fragmentation chain transfer (RAFT).[2] RAFT polymerization[46] has been successfully applied to grow well-controlled brushes on polymer surfaces to alter the surface properties of the material used such as microspheres,[7] cellulose[8] or a cross-linked polymer disk.[9] RAFT polymerization has been employed to generate well-dened brushes on surfaces[1013] or to obtain inorganic particles with controlled polymer hair size.[14,15] The inorganic material investigated using the RAFT process for grafting includes gold,[11,15] silica/ silicon[10,12,14] and polytetrauoroethylene (PTFE) surfaces[13] The RAFT agents can be immobilized on the surface[8,12,14,15] or radicals can be generated on the surface by covalent attachment of an azoinitiator while the RAFT agent has been added to the solution.[10,13] The immobilization of the azoinitiator on the surface with free RAFT agent in the solution was reportedly only successful when free azoinitiator was added to the system.[16] It is, therefore, advantageous to immobilize the RAFT agent directly on the surface. Two methods are available: the RAFT agent can either be attached via the R-group or the Z-group of the RAFT agent.[4,5] However, attachment via the R-group will always lead to detachment of the RAFTagent during the polymerization, and controlled growth of brushes is only expected for very low conversion.[14] This study is to the best of our knowledge the rst investigation of the immobilization of the RAFT agent via the Z-group on a at surface. Perrier et al used the Z-group approach to grow well-controlled polymers from silica particles and Merrield resins.[17] We have exploited this route to prepare well-dened biocompatible and temperature-responsive polymer brushes. With the attachment of the RAFT agent via Z-group we expect that no thiocarbonylthio group will be lost during the course of the polymerization since the RAFT agents always stay covalently attached close to the surface. The connection of RAFT agent via Z-group was applied earlier as a possible pathway to prepare comb polymers from soluble cellulose.[18] We will demonstrate here that the Z-group attachment approach leads to the controlled growth of block copolymer brushes, where the second block grows underneath the rst block. Control is demonstrated by the synthesis of glycopolymer brushes on surfaces. Such brushes are important because of the signicant role glycopolymers play in specic recognition events in cell-cell communication. Chain extension of the surface using N-isopropyl acrylamide (NIPAAm) cannot only conrm the livingness of the process but will
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also result in the formation of stimuli-responsive glycopolymer brushes (Scheme 1).

Experimental Part
Materials The synthesis of N-acryloyl glucosamine (AGA),[19] 3benzylsulfanylthiocarbonyl sulfanylpropionic acid (I) and 3benzylsulfanylthiocarbonyl sulfanylpropanoyl chloride (II) was prepared as described previously.[20] 4,40 -Azobis(cyanopentanoic acid) (ACPA, Fluka, 98%), N-isopropylacrylamide (NIPAAm) (Aldrich, 97%), 3-aminopropyl trimethoxysilane (APTMS) (Aldrich) were used without further purication. All solvents were HPLC grade (Asia Pacic Specialty Chemicals). Synthesis Pre-cut silicon pieces (%1.0 1.0 cm2) were rinsed with ethanol, acetone and then immersed in piranha solution (conc. H2SO4/H2O2) for approximately 1 h. The silicon substrates were then washed thoroughly with deionized water. After a washing step with ethanol the wafers were blown dried and immediately immersed in a solution of APTMS (1 vol.-%) in toluene. The samples were heated for 1 h at 60 8C[21] followed by rinsing with toluene, acetone, and ethanol. After drying the silicon pieces were immersed in a solution of 3-benzylsulfanylthiocarbonyl sulfanylpropionoyl chloride in dry chloroform (5 vol.-%). The mixture was left to react over night. The samples were then rinsed with chloroform and ethanol and stored in a dark container to avoid decomposition of the RAFT agent via the inuence of light. Grafting of AGA was performed using a treated silicon waver with immobilized RAFTagent immersed in a solution of AGA (0.715 mol L1), 4,40 -(azobis(cyanopentanoic acid) (7.14 104 mol L1) as the initiator and the trithiocarbonate agent (I) as RAFT agent (3.57.103 mol L1) in deionized water-ethanol mixture (5:1 v/v). After degassing via freezepump thaw cycles the reaction vessels were immersed in a water bath at 60 8C for a preset time. A monomer conversion of 65% was achieved after a reaction time of 6 h. The polymer brushes were puried with sufcient rinsing with water and additional immersion in water under ultrasonication. Grafting of NIPAAm brushes was performed by immersing a silicon waver (with RAFT agent or PAGA brush) in a solution of NIPAAm (1.82 g, 0.8 mol L1), 4,40 -(azobis(cyanopentanoic acid) (4.0 mg, 7.14 104 mol L1) as the initiator and the trithiocarbonate agent (I) as RAFT agent (10.7 mg, 1.96 103 mol L1) in deionized waterDMSO mixture (1:1 v/v). After a reaction time of 4 h at 60 8C a conversion of 70% was obtained. The polymer brushes were puried with sufcient rinsing with ethanol and additional immersion in ethanol under ultrasonication (see Supplementary Information for more details). Characterization
1

H NMR

1 H NMR spectra were recorded on a Bruker spectrometer (300 MHz) in D2O.

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Scheme 1. Synthesis of stimuli-responsive glycopolymer brushes using RAFT polymerization via Z-group approach.

Gel Permeation Chromatography (GPC) GPC analysis of glycopolymers were performed in N,Ndimethylacetamide (DMAc) (0.03% w/v LiBr, 0.05% BHT) at 40 8C (ow rate 1 mL min1) (more detail on the GPC system can be found in ref.[19]). Contact Angle Measurements Contact angle goniometry was performed using a homemade stage with a computer-controlled microsyringe and digital camera. Infusion and withdrawal rates of 4 mL min1 were used. Static water contact angles (Yst) were recorded. Advancing (Yadv) and receding (Yrec) contact angles were determined via Wilhelmy plate technique using a NIMA DST 9005 computer-controlled Dynamic Surface Tensiometer/ Contact Angle Meter. Brush Thickness Ellipsometric measurements were carried out using a EL X02C ellipsometer from Dr Riss Ellipsometerbau GmbH with a 632.8 nm laser at 708 angle of incidence. Refractive indices of 1.36[22] and 1.45 were used for PNIPAAm and PAGA, respectively.
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Results and Discussion

The attachment of the RAFT agent via Z-group or R-group can considerably inuence the outcome of the grafting of polymer brushes. R-designed RAFT agents allow the termination of two macroradicals on the surface resulting in the loss of RAFT agent. In contrast, Z-designed RAFT agents prevent these side reactions. However, the transfer of the macroradical to the RAFT agent is taking place close to the silicon surface. With increasing brush length the thiocarbonylthio group may be less and less accessible and the growing polymer brush has a shielding effect. As observed during the synthesis of star polymers using the Z-group approach this can consequently result in the formation of termination products and the limited growth of the polymer chain.[18,23] The RAFT agent was attached to the surface starting with a known procedure to immobilize 3-aminopropyl methoxy siloxane.[21] The resulting amino-functionalized surface was observed to have a contact angle of 508 (Table 1), which is close to values observed earlier.[21] The thickness of this surface layer of 4 nm indicates, however, that multilayers were obtained, which maybe due to the extended reaction
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Table 1. Polymer brush thickness and contact angle of silicon waver pre-treated with 3-aminopropyl trimethoxy silane (Si-NH2), silicon with immobilized RAFT agent (Si-RAFT), silicon with PAGA and PNIPAAm homopolymer and block copolymer brushes. Sample Reaction time h Si-NH2 Si-RAFT Si-PAGA Si-NIPAAm-PAGA Si-NIPAAm-PAGA Si-PAGA Si-NIPAAm-PAGA Si-NIPAAm-PAGA Si-PAGA Si-NIPAAm-PAGA Si-NIPAAm-PAGA Si-PNIPAAm Si-PNIPAAm Si-PNIPAAm Dip coated PAGA 2.5 4.0 2.5 4.0 2.5 4.0 2.0 2.5 3.0 Conversion of monomer in solution 0.36 0.75 0.44 0.65 0.38 0.70 0.23 0.35 0.41 Thickness nm 4 5 19 36 49 23 60 66 28 50 75 15 16 27 Static contact angle 8 50 75 73 74 78 78 79 80 68 71 75 55 54 50 48 Adv./Rec. contact angle 8 75/45 82/42 60/40

time. Subsequent reaction with 3-benzylsulfanylthiocarbonyl sulfanylpropanoyl chloride (II) was conrmed by the appearance of an amide bond in the FT-IR at around 1 670 cm1 and the contact angle rises to 758 (Table 1). The RAFT containing silicon wafers were then employed in the homopolymerization of NIPAAm and AGA, respectively. The substrates were immersed in the monomer solution, which contains not only initiator, but also RAFT agent. The added RAFT agent is essential to suppress termination reactions in solution, hence, stabilizing the livingness of the system.[14] PAGA brushes were synthesized in aqueous solution. A small amount of ethanol was required to allow the full dissolution of the RAFTagent. The fraction of water and ethanol in the mixture were carefully composed to accommodate the solubility of the RAFT agent and the glycopolymer. While poly(N-acryloyl glycosamine) is fully soluble in water, the polymer starts precipitating in ethanol. PNIPAAm brushes were prepared employing a mixture of DMSO and water as a solvent. This solvent was shown to fully solubilize PAGA blocks,[19] which is essential for the preparation of block brushes. In addition, the added DMSO altered the LCST of PNIPAAm signicantly and the polymerization at a temperature of 60 8C was therefore possible. During polymerization the radicals are generated in the solution. The growing macroradical either diffuse to the RAFT agent immobilized on the surface or it undergoes chain transfer with free RAFT agent in solution. The RAFT process in solution can be monitored concerning monomer conversion and molecular weight. Both polymerizations, NIPAAm and AGA, proceeded with a pseudo-rst order kinetic plot indicating a constant radical concentration (see Supplementary Information). The rate of polymerization was similar to earlier experiments employing these monomers.[19] Additionally, the molecular weight of the polymer
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in solution increases with conversion close to the theoretical value. To obtain similar molecular weights the monomer to RAFT ratio in the case of NIPAAm was twice as high to accommodate the higher molecular weight of the sugar containing monomer. Two independent experiments were carried out using either NIPAAm or AGA as monomer. The molecular weight was slightly higher than the expected value probably due to the calibration of the GPC system using polystyrene standards. The molecular weight distributions remained narrow throughout the polymerization with polydispersity indices below 1.25 (Figure 1). Both brushes, PNIPAAm and PGA, were found to increase linearly with monomer consumption. The growth of the brush thickness was slightly different when employing NIPAAm or AGA with NIPAAm resulting in slightly thicker brushes. Brushes from 5 to 30 nm were achieved depending on the monomer conversion. At the same conversion, PNIPAAm brushes were typically 5 nm bigger than PAGA brushes despite resulting in the same molecular weight of the polymer in solution. The PNIPAAm polymer brush with a thickness of 26 nm at a monomer conversion of 0.47 corresponds to a molecular weight of the free PNIPAAm polymer in solution of 21 620 g mol1. The grafting density, s, of these chains can be calculated from h MPNIPAAm s rPNIPAAm NA

where h is the thickness of the lm, and NA is Avogadros number. We, therefore, estimate a grafting density of 0.67 chains nm2, which indicates that these brushes are very dense. The linear increase between monomer conversion and brush thickness reveal that the process is controlled. The contact angles of the surface conrmed the alteration of the surface properties with the grafting of PNIPAAm and
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Figure 1. Brush thickness and molecular weight of free polymer in solution of PAGA (squares) and PNIPAAm (circles) homopolymer brushes versus monomer consumption. The solid line indicates the theoretical molecular weight of the polymer in solution calculated using the monomer conversion and the monomer/RAFT agent ratio. The dashed lines represent the best linear t using both independent experiment (open symbol and crossed symbol). (PAGA brushes: [AGA] 0.71 mol L1, [ACPA] 7.1 104 mol L1, [I] 3.5 103 mol L1 in water/ethanol (5:1 v/v); PNIPAAm brush: [NIPAAm] 0.8 mol L1, [ACPA] 7.1 104 mol L1, [I] 1.96 103 mol L1 in water/DMSO (1:1 v/v))

Figure 2. Brush thickness of PAGA-block-PNIPAAm brushes versus NIPAAm consumption. The solid line indicates the growth of the PNIPAAm homopolymer brush from Figure 1. The PAGA brushes employed for chain extension had a thickness of 19 (triangle), 23 (square), 28 (circle) nm. (PAGA-block-PNIPAAm brush: [NIPAAm] 0.8 mol L1, [ACPA] 7.1 104 mol L1, [I] 1.96 103 mol L1 in water/DMSO (1:1 v/v))

PAGA brushes, respectively (Table 1). The contact angles of PNIPAAm brushes were slightly smaller than literature values (Table 1).[24] Additionally, a slight dependency of the contact angle on the layer thickness was observed. This may be simply due to the experimental uncertainties and also due to the arrangement of the brushes depending on their length as reported elsewhere.[13] A signicantly higher contact angle is measured using PAGA brushes compared to a thin PAGA layer prepared by dip coating in a methanol solution. Again, this can be attributed to the different arrangements of polymer chains but also to a visibly reduced surface smoothness of the dip-coated sample. PAGA brushes were subsequently utilized in a chain extension process using NIPAAm to generate stimuliresponsive glycopolymer brushes with block structures. It has to be noted here that the RAFT agent remains in close contact with the silicon surface. Consequently, the NIPAAm will be inserted between the PAGA block and the PNIPAAm and should, therefore, not alter directly the properties of the surface. In earlier studies using a Z-group approach for the synthesis of star polymers[23] this step was reportedly rather difcult. Increasing steric hindrance prevented the macroradical from reaching the RAFT agent, hence, an increasing amount of termination reactions were observed and the branches stopped growing. In contrast here, no signicant differences are observed when comparing the growth of the PNIPAAm brushes with the increase of the thickness of the PNIPAAm block grown from PAGA
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brushes (Figure 2). The PNIPAAm polymer did grow approximately (within errors) at the same rate independent of the initial size of the RAFT agent or the size of the PAGA brush. It is rather surprising to see this signicant difference between the star synthesis using the Z-group approach and the brush synthesis. A possible explanation could be that the macroradical is rather entangled within the brush layer and cannot diffuse away from the reactive site. Consequently, to allow further brush growth, monomer must diffuse into the brush while the polymer chain is entrapped in its position. This mechanism sets our Z-group immobilized RAFT polymerization apart from all other surface-initiated brush growth strategies reported so far. Contact angle measurements conrm the incorporation of the PNIPAAm block between the PAGA block and the surface. No signicant alteration in the wettability of the surface was recorded which could only be explained that the PNIPAAm block is indeed not exposed to the surface. We, therefore, can validate the mechanism of the RAFT polymerization using the Z-group approach as suggested in Scheme 1. For further information dynamic contact angles were determined showing the differences between advancing and receding values (Table 1). The surface hysteresis (difference in the advancing and receding contact angles) has been shown to be a good approximation of the microscale roughness of surfaces.[25] The obtained values reveal a reasonably at surface, but further experiments are required to cor 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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relate these values to exact surface smoothness and subsequently to initiator efciencies.

Conclusion
Stimuli-responsive glycopolymer brushes on silicon wavers were synthesized using the RAFT process. The RAFT agent was attached via Z-group excluding side reactions on the surface such as termination reactions. The brushes grew linear with the monomer consumption in solution conrming a living process. Chain extension reaction of PAGA brushes with NIPAAm reveal that difculties such as steric hindrance resulting in termination reactions as observed in earlier studies are absent or diminished. Contact angle measurement conrmed that the second block is build in between the rst block and the silicon surface.

Acknowledgements: MS would like to thank the University of New South Wales (UNSW) for a John Yu fellowship to support her stay at the Melville Laboratories. MS would also like to thank her colleagues Thomas P. Davis and Christopher Barner-Kowollik for their support, which made her stay at Cambridge possible. MS would also like to thank the members of the Melville lab for their warm welcome.

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