Surface Modification of Poly (divinylbenzene) Microspheres via

Surface Modification of Poly (divinylbenzene) Microspheres via

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Surface Modification of Poly(divinylbenzene) Microspheres via Thiol-Ene Chemistry and Alkyne-Azide Click Reactions

Anja S. Goldmann,† Andreas Walther,† Leena Nebhani,# Raymond Joso,‡ Dominique Ernst,§ Katja Loos,| Christopher Barner-Kowollik,*,⊥ Leonie Barner,*,# and Axel H. E. Muller*,†

Makromolekulare Chemie I and Zentrum fur Kolloide and Grenzflachen, UniVersitat Bayreuth, 95440 Bayreuth, Germany, Centre for AdVanced Macromolecular Design, School of Chemical Sciences and Engineering, The UniVersity of New South Wales, Sydney, NSW 2052, Australia, Experimentalphysik IV and Bayreuther Institut fur Makromolekulforschung (BIMF), UniVersita¨t Bayreuth, 95440 Bayreuth, Germany, Department of Polymer Chemistry & Zernike Institute for AdVanced Materials, UniVersity of Groningen, 9747AG Groningen, The Netherlands, PreparatiVe Macromolecular Chemistry, Institut fur Technische and Polymerchemie, UniVersitat Karlsruhe (TH)/ Karlsruhe Institute of Technology (KIT), Engesserstrasse 18, 76128 Karlsruhe, Germany, and Fraunhofer Institut fur Chemische Technologie, Joseph-Von-Fraunhofer-Strasse 7, 76327 Pfinztal (Berghausen), Germany

ReceiVed February 13, 2009; ReVised Manuscript ReceiVed March 31, 2009

ABSTRACT: We report the functionalization of cross-linked poly(divinylbenzene) (pDVB) microspheres using both thiol-ene chemistry and azide-alkyne click reactions. The RAFT technique was carried out to synthesize SH-functionalized poly(N-isopropylacrylamide)(pNIPAAm) and utilized to generate pNIPAAm surface-modified microspheres via thiol-ene modification. The accessible double bonds on the surface of the microspheres allow the direct coupling with thiol-end functionalized pNIPAAm. In a second approach, pDVB microspheres were grafted with poly(2-hydroxyethyl methacrylate) (pHEMA). For this purpose, the residual double bonds on the microspheres surface were used to attach azide groups via the thiol-ene approach of 1-azido-undecane-1-thiol. In a second step, alkyne endfunctionalized pHEMA was used to graft pHEMA to the azide-modified surface via click-chemistry (Huisgen 1,3-dipolar cycloaddition). The surface-sensitive characterization methods X-ray photoelectron spectroscopy, scanning-electron microscopy and FT-IR transmission spectroscopy were employed to characterize the successful surface modification of the microspheres. In addition, fluorescence microscopy confirms the presence of grafted pHEMA chains after labeling with Rhodamine B.


In recent years, grafting techniques have been employed to affect the attachment of polymers onto surfaces of nano- and microparticles.1,2 Surfacemodificationof microspheresto obtain shell-functionalized microspheres is an interesting tool for modifying their properties.3 Various approaches toward the surface-modification of poly(divinylbenzene) microspheres (pDVB) have been published over the past years. In general, two different approaches can be categorized, the “grafting to” and the “grafting from” approach. Several groups chose the “grafting from” technique because it allows growing polymer chains from the initiators on the substrate, leading to high grafting densities because the monomer units can easily diffuse to the propagating sites. Various living/controlled free polymerizations techniques can be employed for this purpose, e.g. the reversible addition-fragmentation chain transfer (RAFT) process or atom transfer radical polymerization (ATRP). In the “grafting to” technique, the polymer chains carry an active terminal group and are coupled to the active surface. Such an approach allows the characterization of the polymer chains before coupling but tends to suffer both from low grafting rates4 and from low final grafting densities.

The immense amount of scientific interest in “click”- chemsitry in the past yearssin particular for the Huisgen cycloadditionsshows the efficiency and the versatile applicability of this reaction.5,6 The ease of synthesis of the alkyne and azide functionalities, coupled with tolerance to a wide variety of functional groups, stability and reaction conditions,make this coupling process highly attractive for the modification of polymericmaterials.Concomitantly,the thiol-ene reactionmay besundercertainconditionssan efficientway to couplepolymer strands. Therefore, the thiol-ene reaction has started to attract researchers in various areas of material synthesis.7-13 In our laboratories, the copper-catalyzed Huisgen 1,3-dipolar azide/ alkynecycloadditionprocess14-18 as well as the equallyeffective heteroDiels-Alderconjugationchemistries19-21 have been used successfully for a number of efficient coupling reactions.

In addition, several groups have applied the “grafting from” approach for the modification of microspheres. Zheng and Stover reported the ring-opening polymerization (ROP) of ε-caprolactone catalyzed by Al(Et)3 and Al[OCH(CH 3)2]3 from lightly cross-linked poly(DVB80- co-HEMA) micro- spheres22 as well as the grafting of polystyrene from narrow disperse polymer particles by surface-initiated atom transfer radical polymerization. 23 Barner and co-workers employed RAFT polymerization to exert additional control over the design of core-shell pDVB microspheres and functional particles. 19,24,25 Furthermore, Barner and co-workers applied

* Correspondingauthors.E-mail:(A.H.E.M);

(C.B.-K.); (L.B.) or

† Makromolekulare Chemie I and Zentrum fur Kolloide and Grenzflachen, Universitat Bayreut.

# Fraunhofer Institut fur Chemische Technologie. ‡ Centre for Advanced Macromolecular Design, School of Chemical

Sciences and Engineering, The University of New South Wales.

§ Experimentalphysik IV and Bayreuther Institut fur Makromolekulforschung (BIMF), Universitat Bayreuth.

| Department of Polymer Chemistry & Zernike Institute for Advanced

Materials, University of Groningen.

⊥ Preparative Macromolecular Chemistry, Institut fur Technische and

Polymerchemie,Universitat Karlsruhe (TH)/KarlsruheInstitute of Technology (KIT).

10.1021/ma900332d C: $40.75 2009 American Chemical Society Published on Web 04/27/2009 anionic ring-opening polymerization of ethylene oxide to synthesize hydroxyl-functionalized core/shell microspheres.26

Even though “grafting to” techniques can suffer from lower grafting-densities, we demonstrate in here the versatility and success of these two click-techniques via the efficient surfacemodification of pDVB microspheres in combination with controlledradicalpolymerizationtechniques(ATRPand RAFT).

Experimental Section

Materials. 1-Bromo-1-undecanol (98%, Aldrich), methanol

(Merck), tetrahydrofuran(Merck), acetonitrile(Sigma-Aldich),1,4- dioxane (Fisher Scientific), anisole (9%, Sigma Aldrich), dimethylformamide (BDH, Prolabo), CuBr (9,99%, Aldrich), 2-Bromo- 2-isobutyrate,N,N,N′,N′′,N′′-Pentamethyldiethylenetriamine(PMDETA, Aldrich), 2-(Trimethylsilyloxy)ethyl methacrylate (TMS-HEMA, 96%, Aldrich), sodium azide (Sigma-Aldrich), sodium ascorbate (Sigma), N,N′-Dicyclohexylcarbodiimide (9%, Sigma-Aldrich), 4-(Dimethylamino)pyridine (9%, Aldrich), Rhodamin B base (97%, Aldrich), phosphorus oxychloride (98%, Fluka) and copper sulfate (Sigma), tris(2-carboxyethyl)phosphine (TCEP, powder, Aldrich),N-(1-pyrenyl)maleimide(PM, Sigma) were purchasedand used as received.2,2′-Azoisobutyronitrile(AIBN)was recrystallized from methanol. NIPAAm (N-Isopropylacrylamide) was recrystallized from a mixture of benzene and hexane (2:1). The synthesis of the RAFT agent 3-benzylsulfanylthiocarbonylsulfanylpropionic acid (BPATT) has been described elsewhere.27

Synthesis. Synthesis of 1-Azido-undecan-1-thiol. This compound was synthesized by adapting the method by Oyelere et al.

Synthesis of Azido-Functionalized pDVB80 Microspheres

(pDVB-N3). Poly(divinylbenzene) microspheres (pDVB80) were prepared as described by Bai et al.29 (DVB80, which is composed of isomers of DVB (meta and para), 80%, and 3- and 4-(ethylvinyl)styrene 20%, is used for the synthesis of the microspheres). A 1 g sample of pDVB80 microspheres were mixed with 1-Azidoundecan-1-thiol (1 × 10-5 mol) and AIBN (1 × 10-4 mol) in acetonitrile (10 mL) as solvent. The reaction mixture was stirred for 72 h under reflux. The functionalized microspheres were then isolated by filtration through a 0.45 µm membrane and washed thoroughly with tetrahydrofuran, ethanol, and acetone. Soxhlet extraction has been carried out in acetonitrile for5dt o remove any unreacted compounds. The microspheres were dried under vacuum before characterization.

Synthesisof ω-AlkynePoly(HEMA)withATRP(Alkyne-pHEMA).

of the TMS groups. The ATRP of TMS-HEMA in anisole runs as follows: after filtration through a silica column, 53.34 g of TMSHEMA (0.26 mol) monomer was placed in a flask equipped with 164.4 g of anisole, 53.6 mg of CuBr (0.37 mmol), 54.0 mg of 2-propynyl 2-bromo-2-methyl propanoate (0.26 mmol) and a magnetic stirrer bar. The flask was then sealed with a septum and bubbled with nitrogen for 30 min. Then it was heated to 80 °C, and 6 mg of PMDETA (0.38 mmol) was injected under argon to start the polymerization. After 48 h, the reaction was stopped at a conversionof 47.5%. The reactionmixture was purified by filtration over a silica column and dialyzed against THF for 2 weeks.

The cleavage of the TMS protecting groups was carried out by precipitating the p(TMS-HEMA) solution from THF into water in the presence of several drops of concentratedHCl aqueous solution. The white precipitate was freeze-dried from dioxane.

Synthesis of Poly(HEMA) Functionalized pDVB80 (pDVB- g-pHEMA).pDVB-N3 (0.02g) was mixedwithalkyne-pHEMA(0.2 g, 9.5 × 10-6 mol) in dimethylformamide in a Schlenk flask.

Sodium ascorbate (0.19 g, 9.5 × 10-5 mol) dissolved in 1 mL of distilled water was added immediately to the solution. The solution is degassed with nitrogen for 20 min. A degassed flask containing copper sulfate (0.51 mg, 3.2 × 10-6 mol) in distilled water was transferred via a cannula to the Schlenck flask. The solution was stirred for 24 h at 70 °C. Any unreacted compounds were removed by Soxhlet extraction in THF and water.

Synthesis of Rhodamine B Chloride. A solution of Rhodamine

B base (2.5 g, 5.6 mmol) in 1,2 dichloromethane (20 mL)sdried over molecular sieve (3 Å) overnightswas stirred under nitrogen, and phosphorus oxychloride (0.98 mL, 10.6 mmol) was slowly added dropwise over 5 min. The solution was refluxed for 5 h (90 °C). The color turned from dark red to dark purple. Thin layer chromatography (MeOH 100%) indicated full conversion after 4 h. After the dark purple solution was filtered by the use of syringe filters and evaporation of the solvent, the dark purple oily product was dried under vacuum (4.5 mbar) at 45 °C overnight, resulting in a dark-bronze colored solid as a crude product that was not purified further.

Rhodamine B Chloride-Labeling of pHEMA-Functionalized

Microspheres. To fluorescence label the pDVB-g-pHEMA, 1 mg of pDVB-g-pHEMA grafted microsphereswere added to a solution of N,N′-dicyclohexylcarbodiimide as a dehydrating agent (DCC) (5.0 mg, 5.8 × 10-6 mol), 4-(dimethylamino)pyridine (1.0 mg, 8.2 × 10-6 mol) and Rhodamine B acid chloride (5.2 mg, 1.1 × 10-5 mol) in 2 mL THF. The degassed mixture was stirred for 24 h at room temperature. Particles were washed thoroughly with THF, water and ethanol. As a control experiment, pDVB80 microspheres were submitted to the same reaction conditions.

Synthesis of pNIPAAm45. In a round-bottom flask, 4.53 g of NIPAAm (40 mmol), 242.1 mg of 3-benzylsulfanylthiocarbonyl- sulfany propionic acid (BPATT, 8.9 × 10-4 mol), and 72.9 mg (4.5 × 10-4 mol) of AIBN were dissolved in 27 mL of dioxane. The flask was sealed with a rubber septum and the solution was degassed by nitrogen bubbling for 20 min. Then the flask was put in an oil bath at 60 °C for 24 h. The polymerization was stopped by cooling the reaction to room temperature under air exposure. The solution was concentrated under vacuum and precipitated in diethyl ether. After filtration the yellow powder was dried overnight undervacuum.A conversionof 82% was determinedby gravimetric measurement. By analysis of the obtained polymer with a NMP SEC, a molecular weight of 5 300 g·mol-1 and a PDI of 1.14 were determined based on a polystyrene calibration.

SH End Group Modification of pNIPAAm45 (pNIPAAm45-SH). Thiol-modification was followed by the procedure published by

McCormickand co-workers.30 To a 50 mL round-bottomflask were further diluted with an additional 15 mL solution of 1 M NaBH4, and the mixture was allowed to react for 2 h. Following reduction, the homopolymer solution was dialyzed against water for3da nd subsequently lyophilized. The resulting dried polymer was then dissolved in DMF, and a solution of tris(2-carboxyethylphosphine) (TCEP) in DMF was added to yield a 150:1 mol ratio of TCEP to polymer. This solution was allowed to react for 24 h, after which it was charged with a solution of N-(1-pyrenyl)maleimide (PM) in DMF to yield a 150:1 mol ratio of PM to polymeric thiol (pNIPAAm-SH).

Thiol-Ene Reaction between pNIPAAm-SH and pDVB80 (pDVB-g-pNIPAAm).pDVB80(0.05g) wasmixedwithpNIPAAm- SH (0.25 g, 4.9 × 10-5 mol) in 10 mL acetonitrile in a Schlenck flask. AIBN (0.025 g, 1.5 × 10-4 mol) was added immediately to the solution. The solution was degassed with nitrogen for 20 min. Subsequently, the solution was stirred for 48 h at 70 °C to ensure complete conversion. Particles were washed thoroughly with acetonitrile and water by Millipore filtration.

3708 Goldmann et al. Macromolecules, Vol. 42, No. 1, 2009

Characterization. NMR Spectroscopy. 1H NMR spectra were recorded on a Bruker ACF300 300-MHz spectrometer.

SEC. These measurements were performed at room temperature on an apparatus equipped with PSS GRAM columns (30 × 8 m, 7 µm particle size) with 100 Å and 1 0 Å pore sizes and a precolumnusing RI (Bischoff)and UV (270 nm, Waters) detection. NMP with 0.05 M LiBr was used as an eluent in the case of pNIPAAm and DMAC in the case of pHEMA. The flow rate was 1.0 mL·min-1 and the WinGPC software was used for evaluation of the obtained data.

X-ray PhotoemissionSpectroscopy.The sampleswere introduced through a load look system into an SSX-100 (Surface Science Instruments)photoemissionspectrometerwith a monochromaticAl KR X-ray source (hν ) 1486.6 eV). The base pressure in the spectrometer during the measurements was 10-10 mbar. The photoelectron takeoff angle was 37°. The energy resolution was set to 1.3 eV to minimize measuring time. Sample charging was compensatedfor by directingan electronflood gun onto the sample. Spectral analysis included a Shirley background subtraction and a peak deconvolution that employed Gaussian and Lorentzian functions in a least-squares curve-fitting program (WinSpec) developed at the LISE, University of Namur, Belgium.

FourierTransformInfrared(FT-IR)TransmissionSpectra.These spectra were recorded using a Bruker IFS 66v/s spectrometer under vacuum at a resolution of 4 cm-1 using the KBr pellet technique. Spectra were recorded and evaluated with the software OPUS version 4.0 (Bruker).

Scanning Electron Microscopy (SEM). SEM images were recorded on a LEO 1530 (Zeiss) instrument, applying the InLens detector with a slow acceleration voltage of 2 kV and sputtering the microspheres with lead to a sufficient material contrast.

Fluorescence Microscopy. The fluorescence microscope (Leica

DMRX) was operated with a HBO lamp as an excitation light source and a filter cube consisting of an excitation bandpass-filter (BP 450-490 nm), a dichroicbeamsplitterwith a cut off wavelength of 510 nm and a detectionfilter (LP 515 nm). With this combination we could observe the emission of the microspheres. We used objectives with several magnifications (20 ×, C Plan; 63 ×, HCX PL Fluotar; 100 ×, PL Fluotar; Leica). For each CCD-recorded frame (ColorViewIII, Soft imagingsystem)we chose an integration time of 50 s for all measured samples.

ConfocalFluorescenceMicroscopy.These images were captured using a Zeiss LSM 510 confocal laser scanning microscope. All images were captured using an oil immersion lens NA 1.3 (Objective Plan-Neofluar 40 × /1.3 oil). Rhodamine B was excited by a 488 nm Argon laser. A main beam splitter (MFT) was used with a long pass filter (488 nm/543 nm). Emission was captured by a spectral detection unit set 560 nm (LP).

Turbidity Study. A titration device, Metrohm automatic 809

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