Spatially Controlled Photochemical Peptide and Polymer Conjugation

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Spatially Controlled Photochemical Peptide and Polymer Conjugation on Biosurfaces Thomas Tischer, Tanja T. Claus, Michael Bruns, Vanessa Trouillet, Katharina Linkert, César RodriguezEmmenegger, Anja S. Goldmann, Sebastien Perrier, Hans G Börner, and Christopher Barner-Kowollik Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm401274v • Publication Date (Web): 15 Oct 2013 Downloaded from http://pubs.acs.org on October 30, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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Spatially Controlled Photochemical Peptide and Polymer Conjugation on Biosurfaces Thomas Tischer†,# Tanja K. Claus,†,# Michael Bruns,§ Vanessa Trouillet,§ Katharina Linkert,‡ Cesar Rodriguez-Emmenegger,†,#,$ Anja S. Goldmann,†,# Sébastien Perrier,+ Hans G. Börner‡ and Christopher Barner-Kowollik†,#,∗ †

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie,

Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76128 Karlsruhe, Germany, *corresponding author: [email protected] #

Institut für Biologische Grenzflächen (IBG), Karlsruhe Institute of Technology (KIT),

Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany §

Institute für Angewandte Materialien (IAM), Karlsruhe Institute of Technology (KIT) and

Karlsruhe Nano Micro Facility (KNMF), Hermann-von-Helmholtz-Platz 1, 76344 EggensteinLeopoldshafen, Germany $

Cell- and Neurobiology, Zoologisches Institut, Karlsruhe Institute of Technology (KIT), Haid-

und-Neu-Str. 9, 76128 Karlsruhe, Germany ‡

Department of Chemistry, Laboratory for Organic Synthesis of Functional Systems, Humboldt-

Universität zu Berlin, D-12489 Berlin, Germany

ACS Paragon Plus Environment

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Key Centre for Polymers & Colloids, School of Chemistry, The University of Sydney, NSW,

2006, Australia ABSTRACT An efficient phototriggered Diels-Alder conjugation is utilized to graft in an effective and straightforward approach poly(trifluoro ethyl methacrylate) (Mn = 3700 g·mol-1, Ð = 1.27) and a model peptide (GIGKFLHS) onto thin hyaluronan films and cellulose surfaces. The surfaces were functionalized with an o-quinodimethane moiety – capable of releasing a caged diene – via carbodiimide mediated coupling. The o-quinodimethane group is employed as a photoactive linker to tether predefined peptide/polymer strands in a spatially controlled manner onto the biosurface by photoenol ligation. An in-depth characterization employing XPS, ToFSIMS, SPR, ellipsometry and AFM was conducted to evidence the effectiveness of the presented approach. KEYWORDS Surface modification, Cellulose, Hyaluronic acid, Photoconjugation

Introduction Cellulose is one of the most abundant biopolymers – with an annual production of up to 1012 t – playing a vital role in industrial applications and as a starting material for numerous chemical modifications.1-3 Therefore, cellulose is a well characterized substrate and the present –OH moieties can be readily modified to alter the properties and/or introduce entities with specific functions. Cellulose is today obtained from one of the following three sources: wood, cotton or bacteria.4 In recent years different morphologies were increasingly placed into the focus of research such as microfibrillated5,6 or nanocrystalline7 structures.


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Hyaluronic acid (HA) – hyaluronan, an immunoneutral polysaccharide ubiquitous in the human body8 – is of increasing interest with applications in personal care products and in biomedicine, for instance to develop tissue surrogates.9 Since HA is water soluble it plays a vital role in biological processes.10 For instance, it is a crucial element in embryonic development, tissue support-organization, wound healing,11 angiogenesis, tumorigenesis,12 and possibly even in the biomechanical properties of tissues. In addition, HA associates with receptors at the cell-surface which regulate cell motility and adhesion.13-15 It is worth noting that HA is rapidly turned over in the body by hyaluronidase with half-lives raging from hours to days.16 Thus, it holds great promise in the development of engineered tissues and biomaterials for a variety of applications ranging from cardiovascular, orthopedic, pharmacologic and oncologic. It has been the subject of a broad field of research activities since its discovery in 1934 by Meyer and Palmer,17 and became one of the few polymers transferred to clinical usage.

9,13,18-20

Most of the applications

employ HA as a (medical) device exploiting its physical properties, while neglecting the chemical modification opportunities it offers.21 Thus, the modification and alteration of the chemical properties of HA will increase the applicability of this renewable biosubstrate. Chemically, HA represents a glycosaminoglycan featuring three hydroxy (two secondary, one primary), one carboxy and one amide moiety, which are readily accessible for modification purposes. The modification of various surfaces with HA has been achieved via several approaches:9,13,15,21 For example, Turner and co-workers demonstrated that the underlying (or base) substrate influences the ability of HA films to immobilize ovarian cancer cells.22 Bhakta et al. immobilized a bone morphogenetic protein to enhance fracture healing while preventing the severe side effects of the protein.23 Suh et al. demonstrated a technique to pattern diverse


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substrates with HA applying a molding and printing approach.24 It is also possible to build covalent linked layers of HA on substrates. These 2-dimensional scaffolds are of great interest for cell therapy and to study various biological mechanisms in cells to design novel biomaterials. For this HA needs to be covalently bound to a substrate and subsequently functionalized with specific biological cues, such as peptides, growth factors, proteins, etc. To generate monolayers of HA on surfaces, Barbucci and co-workers utilized amino-silane chemistry.25 They investigated the interaction of these layers with biological environments and could demonstrate resistance to non-specific cell-adhesion, while Yue et al. described the functionalization of polydimethylsiloxane (PDMS) surfaces with HA and HA-Collagen conjugates.26 The structure of HA materials was also exploited in 3D by using hydrogels that were functionalized with antibodies to capture cells.27 Langer and co-workers demonstrated that it is further possible to coat glass or silicon surfaces with HA and that these films remain stable after treatment with phosphate buffer saline (PBS) solution for 7 days.28 Patterning biochemical cues in HA-based materials is essential to engineer microenvironments for cells and design biomaterials with precise functions. Various approaches have been utilized to functionalize this versatile polymer for tissue engineering,9 however most of them lacked the ability to spatially control the incorporated moieties. Photopatterning HA utilizing orthogonal approaches emerges as a possible avenue to prepare novel HA biomaterials. A recent approach from Gramlich et al. utilized photopatterning of HA hydrogels employing thiol-norbornene chemistry.29 Although their approach allowed to immobilize thiol functionalized peptides, it required the addition of a photoinitiator to generate radicals. Compared to other methods based on the direct photoactivation of chemical groups at the surface, the approach of Gramlich et al. features limited spatial control and can lead to side reactions. Thus, new methods to photo-pattern


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surfaces are of critical importance if an effective translation of this biopolymer to tissue engineering applications and clinical practice is desired. To functionalize biosurfaces, a robust and highly effective conjugation technique is required to achieve high grafting densities and allow for a controlled and uniform functionalization. Furthermore, the reaction sequence should avoid toxic metal catalysts and harsh reaction conditions. For surface functionalizations, two main approaches are commonly employed, ‘grafting-to’ and ‘grafting-from’. In the ‘grafting-to’ approach, predefined macromolecules are grafted onto a suitable functionalized surface allowing a detailed analysis of the grafted entities prior to their attachment to the substrate.30-32 Especially when employing biomolecules - where it is extremely important to have a well-defined chemical structure and conformation - the ‘grafting-to’ approach is superior to the 'grafting-from' technique. ‘Grafting-from’, on the other hand, builds the polymer or peptide strands directly from a suitable initiation moiety on the surface.33-36 Currently, only a small number of biosubstrates have been functionalized with photoactive moieties. Li et al. synthesized hyaluronic acid–graphene oxide conjugates as photosensitizer for cancer targeted photodynamic therapy.37 Chiono et al. were able to stimulate hydroxyapatite deposition on chitosan-g-fluorescein conjugates.38 As reported previously by our team, the modification of cellulose with tetrazole as photoactive moiety showed excellent properties for the spatially controlled functionalization of cellulose.39 In the current study a phototriggered o-quinodimethane (photoenol) chemistry was selected for the spatially resolved encoding of cellulose and HA surfaces, as it proceeds under very mild reaction conditions, is rapid and requires no catalyst. Other photochemical conjugation reactions


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such as photolabile protection groups (i.e. 2-nitrobenzyl),40 oxime ligations,41 thio-norbornene29 or benzophenone moieties42 lack the orthogonality required for a modular functionalization approach. We herein present an approach, where thin HA films covalently bound to a modified Si surface and cellulose surfaces (filter paper) are functionalized with synthetic polymers and a model peptide (GIGKFLHS) in a spatially resolved fashion employing photoenol chemistry (Scheme 1). The selection of a synthetic polymer and a peptide to demonstrate the power of this approach correlates with the need to engineer the physico-chemical properties of the biomaterials’ interface by changing its surface energy, topography and chemical nature (by utilizing polymers) and to generate niches with specific cues for cells to proliferate and differentiate (by utilizing peptides or other biomacromolecules). The introduced strategy is thus two-pronged: on the one hand, we develop the spatially resolved modification of surface-bound HA, while on the other hand we employ the same technology with cellulose to demonstrate the versatility of the photo-enol-driven ligation to prepare biofunctional surfaces with synthetic polymer strands and biomolecules. The tethering of HA onto the Si surface was characterized in detail via ellipsometry, surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). The success of the spatially resolved surface functionalization is evidenced by XPS as well as ToF-SIMS experiments. The current study is methodologically driven to establish spatially resolved grafting procedures on biosubstrates and thus does not focus on the properties of the obtained materials. Nevertheless, the spatially controlled functionalization of biosubstrates via the employed techniques serves as a proof-of-principle for the design of spatiotemporally controlled functionalization of biosubstrates. It is envisioned that the research


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presented herein can be readily extended towards the design of functional biomaterials for tissue engineering and the biomedical practice.


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Experimental Section Materials Methyl

4-((2-formyl-3-methylphenoxy)methyl)benzoic acid43

tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl

and

2-(1,3-dioxo-3a,4,7,7a-

2-bromo-2-methylpropanoate44

were

synthesized according to literature. Hyaluronic acid functional Si wafers were synthesized according

to

literature

utilizing

Si

surface

instead

of

PDMS

and

employing

aminopropyltriethoxysilane.26 All other chemicals were used as received.

Synthesis of photoenol functionalized hyaluronic acid films Spincoating approach Pretreatment of Si wafers: Single wafers 1×1 cm² were dispersed in Caro’s acid (4 mL H2SO4/H2O2 (3:1)) and allowed to stand for 4 h. Caution: Caro’s acid is HIGHLY corrosive, handle with care and apply suitable safety measures. The Si wafers were rinsed with deionized water. After activation the Si surface presents a layer of silicon oxide, which can be readily functionalized. Hyaluronic acid sodium salt was dissolved in deionized water (4mg·mL-1) and spin-coated on a Si wafer with 800 rpm for 80 s. Subsequent functionalization corresponding to (3).

Covalent tethering approach on hyaluronic acid Amine-functionalization of Si wafers Pretreatment of Si wafers: All Si wafers were cleaned three times by ultrasonification for 15 min in chloroform, acetone and ethanol. Preactivation of the surfaces was achieved by plasma cleaning. After activation of the Si surface, a layer of silicon oxide is formed, which can be


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readily functionalized. Preactivated substrates were placed separately in small glass vials containing a solution of triethoxy aminopropylsilane (10 µL) in 2 mL toluene. The wafers were subsequently heated to 50 °C for 2 h without stirring. The solution was brought to ambient temperature and the wafers were left immersed for an additional 16 h. The wafers were subsequently ultrasonicated in dry toluene (10 mL, 10 min), acetone (10 mL, 5 min) and chloroform (10 mL, 5 min) to remove any physisorbed silane and subsequently dried under a stream of nitrogen.

Hyaluronic acid tethered on Si wafers (HA-Si) (1) EDC·HCl and NHS were added to a HA solution (0.6 mg mL·L-1) in MES buffer (5mmol·L-1, pH = 5.0) to a final concentration of 2.0 mg mL·L-1, respectively. The solution was subsequently stirred at RT for 30 min, after which time the aminated Si samples were loaded (1 mL per sample). The mixture was agitated on a shaker at ambient temperature overnight, and the HA grafted substrates were washed with deionised water and MES buffer.

Photoenol functionalized HA-Si (PE-HA-Si) (2) 4-((2-formyl-3-methylphenoxy)methyl)benzoic acid (10 mg, 0.037 mmol) and diisopropyl carbodiimide (0.048 mL, 0.310 mmol) were dissolved in 2 mL DCM in a sample vial with a septum. The pretreated Si wafer with HA was immersed in the solution. The solution was allowed to shake for 16 h. The Si wafer was then immersed in 10 mL CH2Cl2 and allowed to shake for 30 min. The solvent was renewed and the wafer was shaken for another 15 min. Finally, the wafer was air dried.


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Synthesis of photoenol functionalized cellulose Pre-treatment of Cellulose (1’)30 Filter paper samples (Whatman No. 5) were standardized employing a hole puncher to generate circles with a diameter of 1 cm. 0.4 g of cellulose (20 pieces) was suspended in an aqueous solution of 10 wt% NaOH (150 mL) and kept on a shaker overnight. The basic cellulose pieces were washed with ethanol until a neutral solution was obtained. The swollen cellulose sheets were stored under ethanol and used directly in further reactions after solvent exchange.

Photoenol functionalized Cellulose (Cel-PE, 2’) For solvent exchange, pre-treated Cellulose (1’, 1 piece) was placed in CH2Cl2 and subsequently placed in a solution of methyl 4-((2-formyl-3-methylphenoxy)methyl)benzoic acid (PE, 20 mg, 0.074 mmol, 1 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 28.7 mg, 0.195 mmol, 2.5 eq) and 4-(dimethylamino)-pyridine (DMAP, 2.3 mg, 0.002 mmol, 0.25 eq) in 2.5 mL anhydrous CH2Cl2. The solution was flushed with nitrogen for 2 min and shaken at ambient temperature overnight. The product was washed for 20 min with 10 mL CH2Cl2, 10 mL THF, 10 mL deionized H2O and 10 mL THF, respectively, and was subsequently stored in THF.

Synthesis of maleimide functional PTFEMA (MI-PTFEMA) In a 20 mL Schlenk tube, equipped with a stirring bar, 2,2,2-trifluoroethyl methacrylate (TFEMA, 2.94 g, 17.5 mmol, 40 eq), N,N,N′,N′,N′′-pentamethyldiethylenetriamine (PMDETA, 19.1 mg,

0.11 mmol,

0.25 eq),

Cu(II)Br2

(1.2 mg,

0.006 mmol,

0.0125

eq),

and

2-(1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl-2-bromo-2-methylpropanoate (313.3 mg, 0.88 mmol, 2 eq) were dissolved in 2 mL anhydrous toluene. The tube


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was sealed with a stopper and three freeze-pump-thaw cycles were employed to remove oxygen from the solution. Next Cu(I)Br (6.6 mg, 0.05 mmol, 0.105 eq) was added while the solution was frozen under a nitrogen atmosphere and a further freeze-pump-thaw cycle was conducted. The polymerization was carried out in an oil bath at 60°C for 2 h and was quenched by cooling in an ice bath and flushing with air. The reaction mixture was then diluted with THF, passed through a column with neutral Al2O3 in order to remove the copper catalyst and precipitated in ice cold nhexane. Mn,SEC = 3700 g·mol-1, ð = 1.27 (THF SEC, PMMA calibration) Deprotection The furan protected MI-PTFEMA (0.5 g, 0.072 mmol) was immersed in toluene (10 mL) and refluxed for 5 h. Subsequently, the solvent was removed in vacuo resulting in MI-PTFEMA. 1

H NMR (400 MHz, CDCl3): δ[ppm]= 6.74 (s, 2H, CH=CH), 4.34 (s, CH2-CF3), 4.22 – 4.13 (m,

2H, O-CH2), 3.88 – 3.76 (m, 2H, N-CH2), 2.16 – 1.88 (m, -CH2 backbone), 1.82 (s, 6H, -CH3), 1.18 – 0.86 (m, -CH3 backbone).

Synthesis of maleimide functional model peptide GIGKFLHS The peptide synthesis was performed on an Applied Biosystems 433a peptide synthesizer in a 0.25 mmol scale, using a TentaGel standard Rink-amide linker (SRAM) resin as solid support. Fmoc-amino acid derivatives were coupled following standard ABI-Fastmoc protocols (no capping and single couple) in N-methyl-2-pyrrolidone (NMP) facilitated by 2-(1H-benzotriazol1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate

(HBTU)/

diisopropylethylamine

(DIPEA). After final Fmoc removal the resin was transferred to a 40 mL glass reactor, where 3maleimidpropionic acid (MPA) was coupled twice at ambient temperature (Conditions: first coupling:

MPA

(1.25

mmol),

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronoium


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hexafluorphosphate(PyBoP) (1.25 mmol), DIPEA (0.8 mmol) for 6 h. Second coupling: MPA (0.5 mmol), PyBoP (1.25 mmol), DIPEA (0.8 mmol) for 48 h). After each coupling step the resin was washed with NMP. The support was washed finally three times with NMP and dichloromethane (DCM) and dried overnight under vacuum at 25 °C. The peptides were liberated from the resin by treatment with trifluoroacetic acid (TFA)/DCM/triethylsilane(TES) 50:47,5:2,5 v/v% for 2 h to obtain the completely deprotected peptide. The maleimide functionalized peptide was isolated by diethylether precipitation, centrifugation and twice lyophilization from water. The chemical identity of the product was confirmed by ESI-MS analysis showing mass signal at m/z 1008.6 Da, which is assignable to [M+H]+ (Mth, MPA functional GIGKFLHS = 1007.4 Da).

Photoconjugation of the model peptide onto photoenol functionalized biosubstrates 2 or 2’ was placed in a sample holder (see Figure S2) featuring the photo pattern and was transferred to a crimped vial with the model peptide (20 mg, 0.020 mmol). 3 mL dry DMF was added and freed from oxygen via purging with nitrogen under ice cooling for 40 min. The setup was irradiated for 2 h using an Arimed B6 lamp (36 W, emission maximum. 320 nm) from 5 cm distance. The sample was extracted and shaken in 5 mL DMF for 60 min and subsequently for 60 min in 5 mL deionized H2O. Finally, the surface was rinsed with acetone and dried in a stream of nitrogen.

Photoconjugation of MI-PTFEMA onto photoenol functionalized biosubstrates 2 or 2’ was placed in a sample holder (see Figure S2) featuring the photo pattern and was placed in a crimped vial with MI-PTFEMA (20 mg, 0.006 mmol). 3 mL dry DMF was added


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and freed from oxygen via purging with nitrogen under ice cooling for 40 min. The setup was irradiated for 2 h using an Arimed B6 lamp (36 W, emission maximum. 320 nm) from 5 cm distance. The sample was extracted and shaken in 5 mL DMF for 60 min and afterwards for 60 min in 5 mL CH2Cl2 for MI-PTFEMA. Finally, the surface was rinsed with acetone and dried in a stream of nitrogen.

Characterization XPS Investigations were performed on a K-Alpha spectrometer (ThermoFisher Scientific, East Grinstead, UK) using a microfocused, monochromated Al Kα X-ray source (400 µm spot size). The kinetic energy of the electrons was measured by a 180° hemispherical energy analyzer operated in the constant analyzer energy mode (CAE) at 50 eV pass energy for elemental spectra. The K-Alpha charge compensation system was employed during analysis, using electrons of 8 eV energy, and low-energy argon ions to prevent any localized charge build-up. Data acquisition and processing using the Thermo Avantage software is described elsewhere.45 The spectra were fitted with one or more Voigt profiles (binding energy uncertainty: +/- 0.2 eV). The analyzer transmission function, Scofield46 sensitivity factors and effective attenuation lengths (EALs) for photoelectrons were applied for quantification. EALs were calculated using the standard TPP-2M formalism.47 All spectra were referenced to the C1s peak of hydrocarbon at 285.0 eV binding energy controlled by means of the well known photoelectron peaks of metallic Cu, Ag and Au. ToF-SIMS (time-of-flight secondary ion mass spectrometry) was performed on a TOF.SIMS5 instrument (ION-TOF GmbH, Münster, Germany), equipped with a Bi cluster liquid metal primary ion source and a non-linear time of flight analyzer. The Bi source was operated in the “bunched” mode providing 0.7 ns Bi 1+ ion pulses at 25 keV energy and a lateral resolution of


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approx. 4 µm. The short pulse length allowed for high mass resolution to analyze the complex mass spectra of the immobilized organic layers. Images larger than the maximum deflection range of the primary ion gun of 500×500 µm2 were obtained using the manipulator stage scan mode. Negative polarity spectra were calibrated on the C- , C2- , C3- , and Br- or F- peaks. Positive polarity spectra were calibrated on the C+, CH+, CH2+, and CH3+ peaks. Primary ion doses were kept below 1011 ions/cm2 (static SIMS limit). Size Exclusion Chromatography (SEC) measurements were performed on a Polymer Laboratories (Varian) PL-GPC 50 Plus Integrated System, comprising an autosampler, a PLgel 5 µm beadsize guard column (50 × 7.5 mm) followed by three PLgel 5 µm Mixed-C columns (300 × 7.5 mm) and a differential refractive index detector using THF as the eluent at 35 °C with a flow rate of 1 mL·min-1. The SEC system was calibrated using linear poly(styrene) standards ranging from 160 to 6·106 g mol-1 and linear poly(methyl methacrylate) standards ranging from 700 to 2·106 gmol-1. Molecular weights relative to PMMA are reported in the current contribution. Calculation of the molecular weight of poly(methyl methacrylate) proceeded via the Mark-Houwink parameters for poly(methyl methacrylate) (K = 12.8·10-5 dL·g-1 and α = 0.69).48 Electrospray Ionization-Mass Spectrometry (ESI-MS) spectra were recorded on an LXQ mass spectrometer (Thermo-Fisher Scientific, San Jose, CA) equipped with an atmospheric pressure ionization source operating in the nebulizer assisted electrospray mode. The instrument was calibrated in the m/z range 195–1822 using a standard containing caffeine, Met-Arg-Phe-Ala acetate (MRFA) and a mixture of fluorinated phosphazenes (Ultramark 1621) (all from Aldrich). A constant spray voltage of 6 kV was used and nitrogen at a dimensionless sweep gas flow rate of 2 (approximately 3 L·min-1) and a dimensionless sheath gas flow rate of 5 (approximately


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0.5 L·min-1) were applied. The capillary voltage, the tube lens offset voltage and the capillary temperature were set to 10 V, 70 V and 300 °C respectively. The samples were dissolved with a concentration of 0.1 mg·mL-1 in a mixture acetonitrile and water 3:2 containing 0.1 vol.% of acetic acid and infused with a flow of 10 µL·min-1. Ellipsometry measurements were performed on a Spectroscopic Imaging Auto-Nulling Ellipsometer EP³-SE (Nanofilm Technologies GmbH, Germany) in the wavelength range of λ = 398.9−900 nm (source Xe-arc lamp, wavelength step ~10 nm) prior to and after every deposition/modification step. To increase the measurement precision, a 5× objective and position calibrated sample stage was utilized to perform repeated measurements from the same area of 1×2 mm2 on the sample and in this way to exclude the errors from the variations of the layer thicknesses throughout the substrate area. All measurements were performed at angle of incidence AOI = 60°. The ellipsometric data were fitted with multilayer models using EP4-SE analysis software (Accurion GmbH, Germany). The thickness of APTES layer and hyaluronic acid (HA) layers were obtained from simultaneous fitting using Cauchy dispersion function n = An + Bn/ λ2 (APTES: An = 1.413, Bn = 6265 nm2, HA: An = 1.457, Bn = 17500 nm2). The Si/SiO2 substrates were modeled using the dispersion functions published by Herzinger et al.49 Surface Plasmon Resonance (SPR) spectroscopy was conducted on self-assembled monolayers (SAM) of 11-amino undecanethiol (NH2-SAM), which were prepared on gold coated (50 nm) SPR chips. The substrates were rinsed with ethanol and deionised water, blow dried with nitrogen, and cleaned with UV-ozone cleaner (Jelight) for 20 min. The substrates were immediately immersed in a solution of NH2-SAM (1 mM) in ethanol and kept at ambient temperature in the dark for 1 day.50 The covalent immobilization of HA on NH2-SAM was assessed in a model experiment. Initially


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the carboxylic groups of HA (0.6 mg·mL-1 in MES) were activated by addition of EDC (2 mg·mL-1) and NHS (2 mg·mL-1) and stirred for 30 min. The mass HA immobilized was determined by surface plasmon resonance spectroscopy (SPR) using an instrument based on the Kretschmann geometry and spectral interrogation, built by the Institute of Photonics and Electronics, Czech Republic.51 Sensor chips attached to the prism consisted of glass slides coated with titanium (2 nm) and gold (50 nm). The reflected light was collected into an optical fiber and delivered to a spectrometer. Changes in the refractive index at the surface of the gold film were measured by tracking resonant SPR wavelength in the spectrum of reflected light. A flow cell with chambers of 50 µm depth and volume 1 µL was used to confine the sample during the experiment and ensure a fast replacement of the fluids. The measurement was carried out at 25 ºC and controlled within a range of ± 0.01 ºC. Initially, MES buffer was driven through the flow cell by a peristaltic pump until the baseline was achieved. Subsequently a solution of HA was pumped for 3 h, after which it was replaced with MES. The sensor response was estimated as the difference in the resonance wavelength of the surface plasmons in MES before and after contact of the surface with HA solution. From the resonance wavelength shift the mass deposited on the surface can be obtained by calibration. In our conditions, a shift of 1 nm in the vicinity of 750 nm corresponds to a change in adsorbed mass of 150 pg cm-2.50,52,53 The limit of detection (LOD) of this SPR is 3 pg mm-2. Atomic force microscopy was performed on an MFP-3D-BIO AFM (Asylum Research, Santa Barbara, USA) equipped with an electric sample holder and a standard cantilever holder. The electric sample holder was used to ground sample charges by contacting the mica sample surface with a conductive clamp. A silicone HQ:NSC18/No Al cantilever (MikroMasch, Lady’s Island,


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USA) was used in intermittent contact mode (AC-Mode), at 1 µm scan size, 0.5 Hz scan rate and a resolution of 512 or 384 scan lines and points.

Results and Discussion General Strategy In the following, we will initially describe our general strategy for the preparation of coated photoreactive cellulose as well as HA wafers, before proceeding to discuss their chemically induced ligation with a synthetic polymer (PTFEMA) and a model peptide representing a part of the sequence of Magainin I.

Scheme 1. General strategy followed in the present study: Under irradiation with UV-light, the 2-formyl-3-methylphenoxy (FMP) derivative releases – under photo-isomerization – highly reactive caged diene, which can be trapped in a Diels-Alder reaction with a dienophile such as a maleimide.54,55 When the FMP moiety is tethered to a surface, spatially controlled surface modification can be achieved, 43 with either a model peptide sequence or a polymer strand. The utilization of o-quinodimethanes (photoenols) has been shown to allow for the precise and effective patterning of plain Si surfaces.43 In the present contribution, the scope and applicability


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is broadened to far more sophisticated biosubstrates, such as hyaluronic acid and cellulose. On irradiation, the 2-formyl-3-methylphenoxy (FMP) derivative isomerizes to a highly reactive oquinodimethane species, which can be trapped in a Diels-Alder reaction employing maleimides as dienophiles (see Scheme 2).

Scheme 2. General reaction sequence for the photo-isomerization of the FMP derivative and Diels-Alder trapping of the reactive o-quinodimethane intermediate employing a maleimide as dienophile.

Scheme 3 depicts the overall chemical strategy: Hyaluronic acid tethered to a Si surface and cellulose (plain, pretreated filter paper) are modified via carbodiimide mediated esterification with a photoenol moiety. In a subsequent photoconjugation step, the biosurfaces were functionalized in a spatially controlled manner with tailormade peptide and polymer strands. The formation of the HA layer was followed with ellipsometry, SPR spectroscopy and AFM. The functionalized surfaces were characterized employing XPS and ToF-SIMS for an in-depth analysis of the structured surfaces.


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Scheme 3. Synthetic route for the functionalization of HA films and cellulose with tailormade peptides or synthetic polymer strands via photoenol ligation. Reaction conditions: a) 4-((2formyl-3-methylphenoxy)methyl)benzoic acid, DIC/DMAP, CH2Cl2 b) 4-((2-formyl-3methylphenoxy)methyl)benzoic acid, EDC/DMAP, CH2Cl2, c) maleimide functional peptide/polymer, 2 h irradiation (λ = 320 nm), DMF.

Generation of photoreactive biosurfaces In the current contribution, photoenol functionalized hyaluronic acid (PE-HA) films were generated via covalently tethering of HA on amine functionalized Si surfaces (see Scheme 3) employing a modified procedure based on Yue et al.26 The covalent attachment of HA onto a


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solid Si substrate – as opposed to merely spincoating the HA on a Si wafer – results in a much higher stability of the layer. In particular, as most applications of HA coating involved aqueous media, covalent immobilization is an unavoidable step to avoid highly swollen polyanionic chains not to desorb from the surface. In addition, covalent immobilization allows an in-depth and reproducible characterization utilizing surface characterization techniques such as SPR, XPS and ToF-SIMS. We additionally report one example where spin coated HA has been photopatterned (see Figure S3 and Figure S4). The HA films as well as the cellulose surface were functionalized with the photoenol moiety utilizing carbodiimide mediated esterification. The successful HA tethering can be demonstrated via XPS analysis as depicted in Figure 1. The APTES functionalized Si wafers exhibit a pronounced peak for C1s (bottom spectrum in Figure 1) at 285.0 eV, which is associated with the hydrocarbon signal of C-C/C-H. After the covalent tethering of HA, additional peaks are present: In the C1s spectrum (top spectrum in Figure 1) two new species at 288.6 eV (HN-C=O, O-C-O) and 286.8 eV (C-O, C-N) are visible, confirming the attachment of HA on 1.30,56


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Figure 1. C1s XPS spectra of APTES functionalized Si substrate (Si-NH2, Bottom) and HA-Si (1) (Top) illustrating the appearance of N-C=O and C-O species, which can be associated with the introduction of the photoenol moiety. For a better visualization all spectra are normalized to maximum intensity.

The thickness of the prepared layers was determined by spectroscopic ellipsometry. Grafting of HA was homogeneous with thickness of about 7 nm (refer to Table S2). The performance of the HA layer depends on the amount of HA grafted to the substrate. In order to estimate the mass of HA which could be attached to the surface SPR spectroscopy was utilized.50-53 SPR measurement can detect changes in the grafted mass of less than 3 pg·mm-2 in real time, thus allowing to access the extent and kinetics of the covalent immobilization of HA on amino-terminated SAMs. A NH2-SAM was prepared on a gold coated SPR chip and was utilized as a model for the APTES SAM on Si/SiO2. Figure 2 evidences the real time immobilization. A fast immobilization occurs within the first 30 min of the reaction while a plateau is observed after 1 h indicating that negligible amount of HA was immobilized thereafter. In the light of these results, the reaction time must be at least 1 h to


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achieve a maximum HA functionalization. The amount of tethered HA (as the differences between the baselines of MES) was determined to be ∆mHA=236 pg·mm-2. According to the molecular weight of HA stated by the supplier (1.5-1.8·106 g·mol-1), this results in a grafting

HA / MES buffer

800 600 400

MES buffer

-2

MES buffer

density of HA on APTES SAM of 7.9·107-9.5·107 chains·cm-2.

Immobilized HA [pg·mm ]

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200 0 0.0

∆m HA

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Time [h] Figure 2. SPR sensograph of the immobilization of HA on NH2-SAM resulting in ∆mHA=236 pg·mm-2 which corresponds to a grafting density of 7.9·107-9.5·107 chains·cm-2

The homogeneity of the presented immobilization of HA was monitored via atomic force microscopy (AFM). The corresponding images are depicted in Figure 3. On the left hand side the cleaned Si surface after plasma treatment is displayed. The image in the middle shows the aminated surface, whereas on the right hand side the HA tethered on Si surface (1) is depicted.


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Figure 3. Atomic force height imaging micrographs monitoring the immobilization of HA onto Si wafers. Left: cleaned Si surface; Middle: Si substrate functionalized with APTES (Si-NH2); Right: Surface of 1. The AFM measurements clearly evidence – jointly with the ellipsometry and SPR results – that the functionalization of the Si surface with HA proceeds in a homogenous and efficient manner. The subsequent photoenol attachment can be verified via XPS. In Figure 4, the C1s spectra of the HA functional Si surface (left hand side top) and the photoenol functionalized surface (left hand side bottom) are displayed, illustrating a distinctive difference in the intensity of the signal at 285.0 eV, unambiguously confirming the presence of the desired photoactive linker moiety on the HA surface.


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Figure 4. C1s XPS spectra of HA-Si (1, left top), PE-HA-Si (2, left bottom), Cel-OH (1’,right top), and Cel-PE (2’, right bottom). Both bottom spectra exhibit a more pronounced C-C/C-H species at 285.0 eV, evidencing the successful introduction of the photoenol moiety. For a better visualization all spectra are normalized to maximum intensity. To generate the corresponding cellulose-based photoenol surface, a carbodiimide mediated esterification approach was also utilized. As cellulose substrate, pretreated plain filter paper (Whatman No. 5, for details please refer to the Experimental Section) with a high cellulose content was employed to demonstrate the applicability of the conjugation method to a widespread biosubstrate. The resulting surface demonstrates an increase in the signal C 1s at 285.0 eV, corresponding – as above – to C-C/C-H of the introduced photoenol moiety (Figure 4).

Photochemical Grafting of PTFEMA Having prepared the respective photoreactive surfaces, the next step is to employ them in a photo-patterning approach with a synthetic polymer strand. A maleimide functional ATRP initiator was utilized to polymerize trifluoroethylmethacrylate (TFEMA) to generate a well


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defined polymer featuring a reactive ligation point with an easy-to-track heteroatom (fluorine, for the synthetic route please refer to Figure S1). For the photo-conjugation, the HA coated Si wafer has been placed in a sample holder which allows partial irradiation of the surface with UV light in the presence of PTFEMA in solution (cPolymer = 1.8 mM, Mn = 3700 g mol-1, ð = 1.27). Under irradiation with UV light, the o-quinodimethane function isomerizes and forms a reactive diene, which is trapped directly with the MI-PTFEMA in a Diels-Alder cycloaddition. In Figure 5, the XPS spectra of the photoconjugate are depicted. To allow for an XPS analysis of the surface, the substrate was irradiated half (refer to Figure S2 for the setup details). The F1s spectrum of the irradiated part displays a pronounced fluorine signal (3.6 At% refer to Table S1 for detailed surface composition) at 689.1+/- 0.3 eV corresponding to the –CF3 moiety of the attached MI-PTFEMA (shown on the right in Figure 5).57 In the XPS spectrum of the nonirradiated area shown below in Figure 5, no fluorine can be detected, evidencing the spatial patterning achieved by the photochemical process.


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Figure 5. F1s XPS spectra of PTFEMA-g-HA (left) and of PTFEMA-g-Cel (right). Both bottom spectra show the non-irradiated side evidencing that no ligation reaction occurred since almost no fluorine is detectable. The top spectra represent the irradiated side exhibiting a pronounced signal around 689 eV, attributed to -CF3 of the repeating units. For a better visualization all spectra are normalized to maximum intensity.

On the photoreactive cellulose surface of 2’, the same reaction procedure as described for HA was carried out. The presence of the grafted PTFEMA on the cellulose substrate can be visualized again via XPS analysis (refer to Figure 5). The spectrum of the irradiated area exhibits a pronounced signal at 689.1 +/- 0.3 eV corresponding to a –CF3 originating fluorine (8.6 At.%, Table S1) proving the presence of PTFEMA. In summary, with XPS the spatially controlled functionalization with a polymer strand can be confirmed via XPS on both substrates highlighting the modularity of the employed method. While XPS can provide information about the binding situation in irradiated and non-irradiated areas of the two biosubstrates, imaging the spatially controlled functionalization of the substrates requires a more sophisticated shadow mask as well as an analysis technique capable of imaging


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spatial resolution. Thus, to monitor the spatially controlled functionalization, imaging Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) has proven to be an excellent tool.43 Figure 6 shows the mapping of a PTFEMA-g-HA film (left, -CF3- fragment) and the corresponding cellulose substrate PTFEMA-g-Cel (right, F- fragment). The intensity of the signal increases from black (low) to white (high). The clear meander shape visible in yellow unambiguously evidences that only the areas irradiated with UV light exhibit fluorine, originating from PTFEMA. The slightly uneven shape of the meander structure of PTFEMA-g-Cel is caused by the three dimensional fibrous structure of the cellulose substrate hampering the observance of the working distance limit across the imaged 4x6 mm2 area making the ToF-SIMS mapping highly challenging. The ToF-SIMS image provided is to the best of our knowledge the first time a mapping of a spatially controlled grafted polymer strand on cellulose is reported. In an alternative approach, a spincoated HA film was subjected to photo-patterning, employing a film that was – after casting – modified with the photoenol moiety and afterwards under identical reaction conditions as the cellulose and the covalently tethered HA surface (please refer to Figure S3 and Figure S4). Thus, it could be shown that the spincoating approach is suitable for HA modification as well, unless a treatment with water is necessary.


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Figure 6. ToF-SIMS images of PTFEMA-g-HA (tethered, left) and PTFEMA-g-Cel (right): mapping of –CF3 (for HA, left) and –F (for Cellulose) corresponding to TFEMA monomer unit shows clearly the spatially controlled functionalization of the PE-HA-Si film and the Cel-PE substrate.

Photochemical Grafting of a Model Peptide Sequence The results of XPS and ToF-SIMS analysis reveal the ability to graft polymer chains spatially controlled on HA films and cellulose surfaces. The development of functional biointerfaces requires that the surface interacts with surrounding biological entities. In particular, cells sense their surface by interaction of proteins on their membrane. This information can be encoded using small synthetic peptides. A small sequence can give information for cells to adhere, grow, proliferate or enter apostosis. Thus, in a subsequent step, a model peptide was employed composed of a short sequence (GIGKFLHS, for the complete sequence information please refer to Scheme 3) of the peptide Magainin I. Magainin I is reported to display antibacterial properties not only in solution, yet also in its bound state.58,59 The model peptide bears, similar to PTFEMA, a maleimide moiety as a terminus. After photo-ligation on 2 (HA surface (half


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irradiated)), the XPS analysis indicates the expected increase of the 285.0 eV signal corresponding to C-C/C-H in the irradiated area (see Figure 7), since the model peptide exhibits more C-C/C-H than C-O or O-C-O units.

Figure 7. C1s XPS spectra of GIGKFLHS-g-HA (tethered) top: irradiated area; bottom: dark control area. For a better visualization all spectra are normalized to maximum intensity.

Figure 8 depicts the ToF-SIMS images of GIGKFLHS-g-HA where the grafting has proceeded in a spatially resolved fashion using the already employed meander-type shadow mask. On the left hand side a characteristic fragment corresponding to the peptide sequence is depicted (C5H8N3+), whereas the right hand image illustrates the fragment C8H2O- originating from the photoenol moiety and therefore gives a negative image of the peptide layer. Thus, the ToF-SIMS images evidence that a spatially controlled patterning of peptide sequences on HA surfaces is possible. The observation of these fragments further prove that – in the non-irradiated areas – the surface is still intact and, in principle could be utilized in a subsequent step to serve as linker for an additional functionalization of the substrate.


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Figure 8. ToF-SIMS images of GIGKFLHS-g-HA. Left: mapping of C5H8N3+ corresponding to the peptide sequence: Right: mapping of C8H7O- corresponding to a fragment of the photoenol moiety on non-functionalized HA.

The functionalization of the photoenol bearing cellulose (2’) was conducted in the same fashion as for the HA layer. The resulting XPS spectra are depicted in Figure 9. On the right hand side the C1s spectra of the irradiated (top) and non- irradiated area (bottom) are shown. An increase of the C-C/C-H signal at 285.0 eV is visible in the irradiated spectra, proving – as in the case of GIGKFLHS-g-HA – the presence of the peptide. Opposed to the conjugate with HA, on GIGKFLHS-g-Cel the nitrogen signal can be employed to further evidence the presence of the peptide entity on the surface. On the left hand side the N1s spectra of the irradiated area is depicted on the top, the non irradiated control area on the bottom. The pronounced N1s signal at 400.3 eV corresponding to the nitrogen in an amide bond unambiguously proves the presence of the peptide and the success of the photoconjugation.60


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Figure 9. N1s (left) and C1s (right) XPS spectra of GIGKFLHS-g-Cel, top: irradiated area; bottom: dark control area. For a better visualization all spectra are normalized to maximum intensity.

The successful grafting of the model peptide onto Cel-PE could also be illustrated via ToF-SIMS imaging (refer to Figure 10). The left image depicts the CNO- fragment whereas the right image shows a CN- fragment. Since cellulose in general – and therefore Cel-PE as well – lacks nitrogen, these signals represent in combination with the XPS results (refer to Figure 9) the proof of the spatially controlled ligation of the peptide sequence onto the cellulose substrate. Once again, the fibrillar structure of the cellulose leads to a slightly uneven shape of the ToFSIMS image.


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Figure 10. ToF-SIMS images of GIGKFLHS-g-Cel: Left CNO- Right: CN--fragment Both fragments originate from the tethered model peptide.

Conclusions In the present contribution the successful covalent attachment of PTFEMA and peptide strands onto the biosurfaces cellulose and ultrathin HA films has been demonstrated. The formation of the HA films was investigated utilizing ellipsometry, SPR spectroscopy and AFM. The substrates were functionalized with an o-quinodimethane moiety which undergoes isomerization to a reactive diene species when irradiated with UV light. The reactive diene can be trapped in a non-reversible Diels-Alder reaction employing dienophiles such as maleimides. Maleimide functional PTFEMA and a model peptide strand (GIGKFLHS) – a partial sequence of Magainin I having antimicrobial properties – were utilized to modify these biosubstrates in a spatially controlled manner. The resulting interfaces were investigated via XPS and ToF-SIMS imaging to reveal the successful spatially resolved grafting. The introduced photochemical spatially controlled ‘grafting-to’ of peptide strands onto biosurfaces provides a novel and facile avenue to tailor the properties of biomaterials based on cellulose and HA. Thus, it is envisioned that the presented strategy will pave the way for the translation of these and other biomaterials from laboratory to clinical practice.


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Acknowledgements C. B.-K., A.G. and H. B. acknowledge financial support for the current project from the German Research Council (DFG). In addition, C. B.-K. acknowledges funding from the Karlsruhe Institute of Technology (KIT) in the context of the Helmholtz BioInterfaces program. T.T. acknowledges funding from the Karlsruhe House of Young Scientists (KHYS) supporting a research stay at the University of Sydney, Sydney, Australia. C.R.-E. thanks the Alexander von Humboldt Foundation for financial support via Humboldt Research Fellowships for Postdoctoral researchers and the Grant Agency of the Czech Republic (GACR) under Contract No P205121702. The authors thank Alexander Quick (KIT) for his support with surface activation, Peter Krolla-Sidenstein for the AFM measurements and Ognen Pop-Georgievski from the Academy of Sciences of the Czech Republic for the ellipsometry measurements.

Supporting Information Available: Elemental analysis, Ellipsometry results, C1s spectra, TOF-SIMs images, molecular weight distribution, and ESI-MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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Table of contents (ToC) graphic

Spatially Controlled Photochemical Peptide and Polymer Conjugation on Biosurfaces

Thomas Tischer, Tanja K. Claus, Michael Bruns, Vanessa Trouillet, Katharina Linkert, Cesar Rodriguez-Emmenegger, Anja S. Goldmann, Sébastien Perrier, Hans G. Börner and Christopher Barner-Kowollik*


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Spatially Controlled Photochemical Peptide and Polymer Conjugation

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