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One-step fabrication of biocompatible multifaceted nanocomposite gels and nanolayers Fuat Topuz, Matthias Bartneck, Yu Pan, and Frank Tacke Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01483 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 20, 2016

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One-Step Fabrication of Biocompatible Multifaceted

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Nanocomposite Gels and Nanolayers

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Fuat Topuza,b* Matthias Bartneck,c Yu Pan,d and Frank Tackec

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a

DWI-Leibniz Institute for Interactive Materials e.V., RWTH Aachen University, Forkenbeckstrasse 50,

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52074 Aachen (Germany)

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b

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c

UNAM-National Nanotechnology Research Center, Bilkent University, 06800 Ankara (Turkey)

Department of Medicine III, Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, 52074

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Aachen (Germany) d

Biomedical Engineering, Biointerface Laboratory, RWTH Aachen University, 52074 Aachen (Germany)

* To whom correspondence should be addressed: Dr. F. Topuz; E-mail. [email protected]

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ABSTRACT. Nanocomposite gels are a fascinating class of polymeric materials with an integrative

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assembly of organic molecules and organic/inorganic nanoparticles, offering a unique hybrid network

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with synergistic properties. Mechanical properties of such networks are similar to those of natural

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tissues, which make them ideal biomaterial candidates for tissue engineering applications. Existing

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nanocomposite gel systems, however, lack many desirable gel properties and their suitability for surface

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coatings is often limited. To address this issue, this paper aims at generating multifunctional

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nanocomposite gels that are injectable with an appropriate time window, functional with bicyclononynes

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(BCN), biocompatible, slowly degradable, and possessing high mechanical strength. Further, the in situ

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network forming property of the proposed system allows the fabrication of ultrathin nanocomposite

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coatings in the sub-micron range with tunable wettability and roughness. Multifunctional nanocomposite

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gels were fabricated under cytocompatible conditions (pH = 7.4 and T = 37 oC) using laponite clays,

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isocyanate (NCO)-terminated sP(EO-stat-PO) macromers and clickable BCN. Several characterization

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techniques were employed to elucidate the structure-property relationships of the gels. Even though the

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NCO-sP(EO-stat-PO) macromers could form a hydrogel network in situ on contact with water, the

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incorporation of laponite led to significant improvement of the mechanical properties. BCN motifs with

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carbamate links were used for a metal-free click ligation with azide-functional molecules, and the

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subsequent gradual release of the tethered molecules through the hydrolysis of carbamate bonds was

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shown. The biocompatibility of the hydrogels was examined through murine macrophages, and it has

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shown that the material composition strongly affects cell behavior.

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KEYWORDS. Nanocomposite hydrogels, laponite, click chemistry, macrophages, coatings

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INTRODUCTION. Hydrogels are water-filled swollen polymeric matrices with diverse physical

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properties, which can be engineered to resemble any form. This enables their use as biosensors, cell-

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scaffolds, and drug delivery devices.1-4 However, their mechanical weakness acts as an obstacle in many

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applications, such as tissue regeneration scaffolds in load-bearing tissues, and wound sealants requiring

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higher resistance to an external deformation. Most hydrogels consist of a random arrangement of large

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numbers of covalent cross-links, and thus, are brittle on exposure to an external force because of a poor

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energy dissipation mechanism in the gel network.5,

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incorporation of physical links (e.g., hydrophobic interactions, hydrogen bonding, supramolecular links,

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etc.) can significantly improve mechanical properties and leads to an efficient energy dissipation

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mechanism. Such physical bonds can break and reform dynamically before the fracture of the molecular

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backbone, generating soft and ductile networks with compressibility, stretchability, and recoverability

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due to the presence of intermolecular noncovalent interactions.7-9 In this context, several approaches

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have been developed to improve energy dissipation mechanisms, such as (i) sliding-ring gels, (ii)

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double-network gels, (iii) macromolecular microsphere composite gels, and (iv) hydrogels with

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hydrophobic domains.10-14 Besides, the incorporation of nanoparticles into hydrogels can considerably

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enhance mechanical properties of polymeric networks by acting as additional cross-linking domains.10

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In this regard, silicate-based hybrid gels have shown astonishing properties, which aid in the design of

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novel materials for drug delivery, tissue engineering and biomedical imaging applications due to the

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enhanced surface interaction of polymers with silicate nanoparticles, such as laponites.11

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In addition to the permanent cross-links, the

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+ − [ Mg5.5Li0.3 ) Si8O20 (OH ) 4 ]0.7 Laponites ( Na0.7 ) are compositionally similar to bioactive glasses, and have a

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shape of nanodisk with an average diameter of 25 nm and a thickness of 1 nm.15 They are particularly

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appealing with respect to natural clays due to their excellent uniform dispersibility, and being

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chemically pure and free from crystalline silica impurities.16 Laponite acts as multifunctional physical

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cross-linking points for polymers leading to a hydrogel network. Earlier studies on laponite revealed that


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these particles could be degraded, or dissolved into non-toxic products (Na+, Si(OH)4, Mg2+, Li+) at a pH

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range of 7.3-8.4, resulting in ortho-silicic acid (Si(OH)4) as the major dissolution product.17 Ortho-silicic

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acid can be absorbed by the body and is found in numerous human tissues and organs, such as bone,

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tendon, aorta, liver and kidney.18, 19 Further, a novel study by Khademhosseini and co-workers reported

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that these bioactive nanoparticles can promote the in vitro osteogenic differentiation of human

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mesenchymal stem cells (hMSCs) in the absence of osteoinductive factors, such as bone morphogenetic

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proteins-2 (BMP-2) or dexamethasone.20 Recently, a mix of laponite clays and gelatin was successfully

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implemented in the development of shear thinning nanocomposite systems for hemorrhage treatment

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with a significant decrease in vitro blood clotting times of 77%.21 Besides, laponite clays were employed

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with amino-functional molecules having guanidinium ion (Gu+) pendants to get physically cross-linked

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gel networks.22, 23

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Figure 1. A schematic illustration of BCN-functional biodegradable laponite-NCO-sP(EO-stat-PO)

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gel fabrication. Inset images show; a) injectability and b) patternability of the hydrogel system using a


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PDMS replica (csP(EO-stat-PO) = 10 wt-%, cclay = 1 wt-%). (c) BF-STEM image shows laponite disks on a TEM grid. The implementation of organic or inorganic nanoparticles (e.g., titanate(IV) nanosheets (TiNSs, sodium montmorillonite (NaMMT), and Ag nanoparticles) with polymers in various forms has led to various functional materials for medical and industrial applications.24-31 Existing nanocomposite gel systems are mostly based on mechanically strong hydrogel networks together with biocompatible, responsive or injectable forms.32-36 It remains a great challenge to produce gel networks that combine these properties with a unique functional structure and a nanolayer-forming ability while the network gradually disintegrates over time through hydrolysis. To address this limitation, this work aims at generating nanocomposite hydrogels that are injectable within an appropriate time window, functional with BCNs, biocompatible, slowly degradable, and having high mechanical strength (Figure 1). The presented system can easily yield biodegradable nanocomposite constructs as hydrogels or ultrathin 3D layers in the sub-micron range, depending on a fabrication route, through in situ gelation of precursors. Furthermore, covalently-embedded BCN motifs allow a dynamic alteration of the three-dimensional network structure with a metal-free click reactivity toward azides, which enables the release of the tethered molecules such that the delivery will be dictated by the gradual hydrolysis of carbamate links. EXPERIMENTAL PART Materials. Laponite clay (laponite® RDS) was purchased from Rockwood Additives Ltd. Star-shaped hydroxyl-terminated polyethers with a backbone of 80% ethylene oxide and 20% propylene oxide and a molecular weight of 12 000 g/mol (PDI = 1.15) were obtained from Dow Chemicals (Netherlands), and the hydroxyl terminal groups were functionalized with isocyanate (NCO) groups. Isophorone diisocyanate (IPDI) was purchased from Sigma-Aldrich, and double distilled before use. N-(1R, 8S, 9s)bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl 1,8-diamino-3,6-dioxaoctane (BCN-POE3-NH2) was obtained from Synaffix BV (Netherlands). Azide fluorescent dye (Eterneon™-Azide) was purchased ACS Paragon Plus Environment

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from Jena Chemicals (Germany). Dulbecco's phosphate-buffered saline (calcium and magnesium free, from Sigma Aldrich) was used. The Synthesis of the Isocyanate-Terminated sP(EO-stat-PO). The isocyanate functionalization of the star polymers has been performed as previously described.37 The hydroxyl terminated sP(EO-statPO)s (Mn = 12 000 g/mol; PDI = 1.15) were functionalized with isophorone diisocyanate (IPDI) in a solvent-free process at 50 oC for 4 days under inert atmosphere. Short-path distillation was used to remove the excess of IPDI. Size exclusion chromatography of the product (NCO-sP(EO-stat-PO)) confirmed that no dimer or trimer formation took place after the functionalization. The Fabrication of Multifunctional Nanocomposite Hydrogels. Laponite aqueous solutions were prepared before use at the preset concentrations in PBS under vigorous stirring for at least 1 h. The NCO-sP(EO-stat-PO)s, BCN motifs and an aqueous dispersion of laponite were mixed. Afterwards, the solutions were taken into syringes of 4.7 mm inner diameter, or poured between the plates of rheometer. The mixture was held overnight at 37 oC to complete the cross-linking reactions. During the preparation of hydrogels, the concentrations of both laponite and NCO-sP(EO-stat-PO) were systematically changed. Rheological Analysis. Dynamic rheological measurements were performed with a rheometer (DHR, TA Instruments). Gelation measurements were carried out between the plates (parallel plate, diameter (D) = 40 mm and the gap size is 500 µm) at 37 oC, and during the measurements, a thin film of silicone oil was used to minimize the solvent evaporation. Throughout the measurements, strain (γ) and frequency (ω) were respectively set to 0.1 and 6.28 rad/s to ensure that the oscillatory deformation is within the linear viscoelastic range. Frequency-sweep test was performed in the frequency range of 0.06600 rad/s at strain (γ) = 0.1. Thereafter, strain-sweep test was carried out in the strain range of 0.01-10 at frequency (ω) = 6.28 rad/s. During the measurements, temperature (T) was kept at 25 oC. Stressrelaxation experiments were carried out at various γ.


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Swelling and Degradation Tests. Hydrogel samples were prepared in syringes, and then, immersed in PBS or water at room temperature to reach swelling equilibrium (i.e., one week) by replacing the medium every day. The swelling ratios φ and mrel at the 7th day were calculated as

φ = (D/Do)3

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mrel= (m/mo)

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Where D (or m) and Do (or mo) are the diameter (or mass) of the gel sample at a given time (t) and just after its preparation, respectively. The degradation of the hydrogel samples in PBS at 37 oC was monitored over gel weights after the lyophilization of the samples. Thermogravimetric Analysis (TGA). TGA experiments were conducted using a TG 209 with a TASystem Controller TASC 414/2 from Netzsch. The measurements were performed under nitrogen atmosphere in the range of 25-500 oC with a heating rate of 10 oC/min. Data analysis was performed with Proteus software (ver. 6.1, from Netzsch Instruments). Scanning Electron Microscope (SEM). Hydrogel samples were kept in water for a couple of days, and then, lyophilized. The internal structures of the gels were directly imaged by ultra-high resolution SEM (Hitachi SU9000 at 1 kV) without any sputtering. The EDX analysis of the lyophilized gels was carried out at 15 kV with Hitachi SEM 3000N. Dynamic Mechanical Analyzer (DMA). The mechanical properties of swollen hydrogel samples were examined using a dynamic mechanical analyzer (DMA Q800, TA Instruments) using a submersion clamp. Cylindrical-shaped hydrogel samples were exposed to stress-strain tests with an incremental speed of 0.5 N/min. The compression modulus was determined as the slope of the stress-strain curve at low strains (< 20%). The stress-at-beak (σB) and strain at-beak (εB) were determined by using Universal Analyzer Software (TA Instruments). Wide-Angle X-Ray Scattering (WAXS). WAXS analysis was done using an Empyrean setup from PANalytical. A Cu X-ray tube (line source of 12×0.04 mm2) provided CuKα radiation with λ=0.1542 ACS Paragon Plus Environment

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nm. The Kβ line was removed by a Ni filter. Source and detector moved in the vertical direction around a fixed horizontal sample. After passing a divergence slit of 1/8o and an anti-scatter slit of 1/4o, the beam reached the sample at the center of a phi-chi-z stage. In the Bragg-Bretano geometry used, the beam was refocused at a secondary divergence slit of 1/4o. Finally, the signal was recorded by a pixel detector (256×256 pixels of 55 µm) as a function of the scattering angle 2θ. Subsequently, the peak positions were calculated from q=2π/d=(4π/λ)sinθ, in which q is the scattering vector. The detector was used in a scanning geometry that allowed all rows to be used simultaneously. To reduce the background, the divergent beam perpendicular to the scattering plane was controlled by a 4 mm mask by restricting the width of the beam at the sample position to ca. 10 mm. In addition, the perpendicular divergence was restricted by soller slits to angles ≤ 2.3o. For each measurement, the sample height was optimized. Scans were made with 2θ, the detector axis, moving at twice the rate of the θ-axis of the incident beam. The calibration was checked using a Si reference sample. Differential Scanning Calorimetry (DSC). DSC analysis was performed on a Netzsch 204 differential scanning calorimeter. The freeze-dried gels (~5 mg) were put in closed aluminum crucibles together with an empty crucible as a reference. The samples were cooled down from 25 to -70 oC at a rate of 10 oC min-1, and subsequently warmed up to 25 oC with the same rate. The Fabrication of Nanocomposite Gel Nanolayers. Substrate Cleaning and UV/Ozone Treatment. Glass substrates were first cleaned by sonication in acetone, water and isopropanol 5 minutes for each one. Subsequently, the substrates were dried in a stream of nitrogen. The surface activation was achieved by a UV/ozone treatment for 30 min. After this step, the water contact angles were below the detection limit. The substrates were then used for aminofunctionalization.


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The Aminosilylation of the Glass Substrates. After activation with a UV/ozone treatment, the substrates were treated with 3-aminopropyl-trimethoxysilane (100 µL) in a desiccator at 5 mbar for 1 h. After the removal of 3-aminopropyl-trimethoxysilane, the substrates were kept at 10-2 mbar for one more hour to remove unbound aminosilane compounds. The Fabrication of Ultrathin Hydrogel Coatings. The NCO-terminated sP(EO-stat-PO)s were dissolved in dry THF under an inert gas atmosphere. Then, the solutions were transferred to the clean room. After the addition of laponite dispersions (i.e., prepared in water), they were mixed for one minute, and dropped onto aminosilylated surfaces. A thin hydrogel layer was generated on the aminofunctionalized substrates using a spin-coater by acceleration within five seconds to the final rotation speed at 2500 rpm, and kept rotating for 40 sec. Drug Releasing Study. Azide-modified fluorescence dye (EterneonTM-Azide, Jena Chemicals) was selected as a model molecule for the drug release study. BCN and azide dye were mixed for 2 h to ensure complete reaction, and an aqueous mixture of NCO-sP(EO-stat-PO) and laponite was added afterwards. Once the nanocomposite gel was formed, the release test was carried out in 5 mL PBS solution. At different time intervals, 2 mL solution was taken, and measured by a fluorescence spectrophotometer (Horiba Instruments, Germany) and 2 mL of fresh PBS was replenished. Cell Isolation and Culture. Bone marrow cells were isolated from femur and tibia of eGFP mice that were housed under specific pathogen free conditions as approved by German legal authorities. Bones were perfused using PBS. To obtain fibroblast-conditioned medium (FCM), L929 fibroblasts were cultured in RPMI medium containing 10% fetal calf serum (FCS) for three days. For a generation of bone marrow derived macrophages (BMM), bone marrow cells were cultured in RPMI medium containing 10% FCS and 20% FCM. After three days, 20% of the medium was replaced. When the plates were covered with cells completely, the supernatant was replaced, and one plate of cells was split on four other plates. Cells were cultured in a humidified incubator at 37o C and 5% CO2. For the


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incubation with the materials, 0.2 million cells in one mL RPMI with 10% FCS and 20% FCM were added to the materials. Microscopy. A DMI6000B microscope equipped with a DFC 360FX camera was used (Leica Microsystems, Vienna, Austria). Fluorescence microscopy settings were adjusted based on the fluorescence of the cells on 24-well polystyrene plates after the designated time intervals. Propidium iodide was obtained from R&D systems, Minneapolis, MN, USA. RESULTS and DISCUSSION. Synthesis and Characterization of Multifunctional Nanocomposite Hydrogels. Laponite RDS is a synthetic hectorite-like clay decorated with pyrophosphate ions (P2O74-) leading to negative charges on all faces. Since there are repulsive electrostatic interactions among the clay particles that force them to stay separate even at moderate concentrations, the clays could easily be dispersed in water without forming a gel-like structure. The aqueous solution of laponite RDS has a low viscosity so that it can serve as an injectable dispersion.38 In the phosphate buffered saline (PBS), the aqueous solution of laponite RDS became turbid because of the formation of clay aggregates by ionic interactions. The aggregate formation was confirmed by DLS: The size of the aggregates ranges from 300 to 800 nm with increasing concentration of laponite (Figure 2a). The presence of the clay aggregation was also supported by nonlinear increase of zeta potential values of aqueous laponite solution with the concentration (Figure 2b). After mixing the aqueous clay solution with NCO-sP(EO-stat-PO)s and BCN, functional nanocomposite hydrogels were successfully produced at 37 oC and pH = 7.4. The crosslinking reactions were monitored by the elastic (G´) and viscous modulus (G´´) of the gel solution during the network evolution at low amplitude strains. The formation of BCN-functional NCO-sP(EOstat-PO) hydrogel in the presence and absence of laponite is shown in Figure 2(d, e), where both moduli (G´ and G´´) were recorded over time (t). Terminal isocyanate groups (NCO) are highly susceptible to the hydrolysis, and transform into amine groups (NH2) over unstable carbamic acids. Simultaneously,


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the newly formed amino groups react with the remaining isocyanates, resulting in the formation of a hydrogel network. This cross-linking mechanism was also able to generate microstructured hydrogels using a polydimethylsiloxane (PDMS) replica, on which an aqueous solution of gel components was poured, and left for cross-linking. Subsequently, the cross-linked nanocomposite material was separated from the replica, and a micro-patterned hydrogel structure could be fabricated at a mixture of 10 wt-% sP(EO-stat-PO) and 1 wt-% laponite (Figure 1, inset b). This state clearly demonstrates the suitability of the gel system for patterning applications.

Figure 2. Characterization of aqueous laponite solutions and rheological properties of the functional nanocomposite hydrogels: (a) Hydrodynamic diameters and (b) the zeta potentials of aqueous laponite solutions in PBS. (c) Increasing turbidity of the aqueous laponite solutions in PBS (from left to right). (d) Elastic modulus G´, and (e) the viscous modulus G´´ during the cross-linking of the NCO-sP(EOstat-PO) prepolymers having BCN motifs (0.22 mmol; 0.16 equiv. of star molecule that corresponds to one arm per a sP(EO-stat-PO) molecule) with/without 1 wt-% laponite. Insets show (i) the batch-toACS Paragon Plus Environment

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batch variations of G´, and (ii) gel pictures of the samples with/without 1 wt-% laponite. (f, g) The frequency sweeps of the respective hydrogels. csP(EO-stat-PO) = 10 wt-% and clap = 1 wt-%. During rheological measurements, the gelation temperature (T) was kept at 37 oC. In the absence of laponite, the NCO-sP(EO-stat-PO) prepolymers at 10 wt-% reacted within 20 min, reaching a plateau at elastic modulus (G´) of 1200 Pa and the viscous modulus (G´´) of 7 Pa (Figure 2(d)). However, the addition of 1 wt-% laponite into the polymerization medium, three-fold increase in G´ was observed, demonstrating strong interactions between clays and polymer network. These gelation experiments were repeated three-times, and similar values for G´ were measured (Figure 2d(inset)). Thus, the reinforcement of the sP(EO-stat-PO) networks with laponite incorporation was evident. The hydrogels exhibited frequency independent G´ in the range of angular frequency (ω) from 0.06 to 600 rad/s, suggesting the formation of mechanically strong networks. On the other hand, a significant rise in G'' suggested that clay particles did not only increase gel strength, but also impacted on the toughness of the hydrogel (Figure 2e). The toughness may be associated with energy dissipating properties of the cross-linked network. Tan δ (loss factor, G´´/G´) represents polymer adhesive bonds which are being broken down and reconstituted during dynamic strains relative to those remaining intact and unchanged. The loss factors for both gel systems (i.e., with/without laponite) are respectively found as 0.014 and 0.0058, indicating that extensive rearrangement of highly entangled polymer material took place during strain development.39 Furthermore, the incorporation of laponites into the gelation system induced opaqueness (Figure 2c, inset), implying the presence of laponites as aggregate in the gel matrix. The arrangement and orientation of clays in the gel matrix were studied through wide-angle X-ray scattering (WAXS). Figure 3a displays the XRD patterns of the clays in PBS and a hydrogel matrix, which revealed a broad peak centered on 2Θ of 28o (d-spacing = 3.18 Å) attributable to a low degree of ordering of the clays in both the medium and the gel matrix. However, XRD spectrum of the laponite


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powder displayed sharp peaks at 20o (d-spacing = 4.44 Å), 35.5o (d-spacing = 2.52 Å) and 60o (dspacing = 1.54 Å).

Figure 3. Characterization of the sP(EO-stat-PO) hydrogels. (a) The XRD patterns of the clay dispersion in PBS and the hydrogel with 1 wt-% laponite. Insets show the proposed distribution of clays in the gel matrix, and the XRD pattern of the laponite powder. (b) The AFM micrograph of 1 wt-% laponite dispersion (in PBS) on a mica surface after spin-coating. (c) The DSC curves for lyophilized gels reveal the glass transition temperature (Tg) at -59.6 oC and the crystallization of freezing bound water between -32.3 and -28.8 oC and the corresponding melting at 0 oC. (d) The TGA curves demonstrated the improvement of thermal stability of the NCO-sP(EO-stat-PO) gels with 1 wt.-% laponite.


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An aqueous solution of laponite on a mica surface was analyzed by AFM, which revealed the presence of aggregates of various sizes (Figure 3b). Hence, the formation of gels can be elucidated with the following scenario. First, the laponite forms aggregates in the PBS buffer. Then the cross-linking of NCO-sP(EO-stat-PO)s occurs among the laponite aggregates, leading to an opaque hydrogel network as illustrated as inset in Figure 3a. We also examined the effect of pH on the laponite aggregation. The DLS analyses on an aqueous laponite solution showed a pH-dependent aggregation, where acidic pH values favor micro-sized aggregates in water and PBS (Figure S2 in Supporting Information). To determine the laponite effect on the phase behavior of the sP(EO-stat-PO) gels, DSC analyses were carried out on the lyophilized gel samples with/without 1 wt-% laponite. Linear PEO chains with Mn ≥ 2 kDa are crystalline. Dependent on the cooling rate and molecular weight of PEO, their crystallization and melting temperatures range from 55 to 40 oC and from 58 to 65 oC, respectively.12 The glass transition temperature (Tg) of the amorphous part of PEGs appears at ~ -62 oC. In contrast to the PEG, the poly(EO-stat-PO) is an amorphous polymer, and do not crystallize. The DSC curves of the lyophilized gels are shown in Figure 3c, where a glass transition temperature (Tg) of the laponite-free gel was observed at -59.6 oC with a corresponding heat capacity (∆Cp) of 0.187 J/(g.K) and the melting of bound water at 0 oC (±0.5) with a corresponding enthalpy of 0.96 mW/mg. The incorporation of laponite did not affect Tg, but changed the cold crystallization temperature (Tc) of the bound water. The laponite-free gel showed a Tc at -32.3 oC with ∆H of 0.086 mW/mg. On the other hand, the gel with 1 wt-% laponite displayed a Tc at -28.8 oC with ∆H of 0.186 mW/mg. The thermal behavior of the gels after preparation was investigated by TGA. Figure 3d shows that the TGA curves of the gels displayed a significant mass loss between 50 and 175 oC, related to the loss of water molecules. In the gel with 1 wt-% laponite, the loss of water molecules occurred at higher temperatures as a result of interactions between water and laponite particles. Due to the negative charges on the clay surface, the ionic interactions may exist between water molecules and clay, causing the adsorption of water. The water molecules in the laponitefree hydrogel release at lower temperatures. The first derivative TGA curves for both samples are shown ACS Paragon Plus Environment

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in Figure S5, where the samples revealed two peaks; the first peak at lower temperatures is related to the water-loss, and the second one to the pyrolysis of the polymer. The pyrolysis of the (sP(EO-stat-PO)) gels started at ca. 300 oC, and ended at 425 oC. The peak pyrolysis temperature shifted from 381 to 390 o

C with laponite incorporation.

Figure 4. Mechanical and swelling properties of the nanocomposite hydrogels. (a) Variation of G´ and tan δ with sP(EO-stat-PO) content. (b) The stress-relaxation profiles of the nanocomposite system (csP(EO-stat-PO) = 30 wt-% and clap = 1 wt-%). (c) Stress-strain curves and (d) strain-at-break values of the 10 wt-% sP(EO-stat-PO) hydrogels with various laponite contents (between 1 and 5 wt-%). Inset shows the compression moduli (Ec) of the gels. (e-f) Relative swelling ratios (mrel or φ) of the hydrogels as a function of laponite concentration in water and PBS. The solid curves are guided to the eyes. The mechanical properties of the nanocomposite gels with 1 wt-% laponite and various sP(EO-statPO) contents are shown in Figure 4a. With increasing sP(EO-stat-PO) concentration, elastic modulus (G´) significantly rose. For example, with an increase of prepolymer content from 10 to 30 wt-%, elastic modulus (G´) rose ten-fold from 3.5 to 35 kPa, while the loss factor (tan δ) decreased from 0.02 to 0.006 ACS Paragon Plus Environment

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so that the network became stiffer. This stiffness can be attributed to the dominating effect of chemically cross-linked polymer network. But, increasing content of laponite leads to tougher networks. Stiffness and the toughness of hydrogels are often inversely related. According to the Lake-Thomas model, a rise in the cross-linking density should decrease the toughness, but increase the stiffness.40, 41 This can be explained by the prominent effect of the cross-linking of the sP(EO-stat-PO)s at high concentrations, which dominates the laponite-induced toughness of the gels. Strain-dependent properties of a nanocomposite gel were measured by stress-relaxation experiments, where the stress σ(t,γ ) was o

recorded after a shear deformation of a controlled amplitude ( γ ) for a duration of 300 s. Figure 4b shows the relaxation profiles of the nanocomposite gel (csP(EO-stat-PO)= 30 wt-%, clap =1 wt-%) at various strains, where the relaxation modulus (Gt) is shown as a function of time (t). The relaxation modulus G(t, γ) decreased with time, and became more distinctive depending on the applied initial strain level. Further, the relaxation process slowly occurred. Interestingly, when the strain (g) rose over 10%, all stressrelaxation profiles first showed a rapid rise in relaxation modulus and then, decrease in modulus (Figure S7). This is due to that the measurements are not anymore in the linear viscoelastic range (LVE). Similar stress-relaxation behavior was observed for the nanocomposite gel prepared at 10 wt-% sP(EO-stat-PO) with 1 wt-% laponite. The mechanical properties of the hydrogels were studied by DMA over the stressstrain curves (Figure 4c, d). Increasing laponite content caused significant changes on the compression modulus (Ec) and the stress-at-break (σB). For example, for an increase in laponite content from 0 to 5 wt-% for the gel with 10 wt-% sP(EO-stat-PO), the compression moduli (Ec) of the respective hydrogels were increased from 2.4 to 120 kPa. For an increase of the sP(EO-stat-PO) concentration from 10 to 30 wt-%, the respective compression modulus of the gel with 1 wt-% laponite rose from 50 to 970 kPa, obviously suggesting the mechanical improvement of the gels with laponite incorporation (in Supporting Information, Fig. S6). These marked changes were also observed in the strain-at-break values (εB) of the hydrogels with incorporation of laponite (Figure 4d). The swelling properties of the hydrogels were


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investigated in water and PBS. Figure 4e and f show equilibrium swelling ratios (mrel and φ) of the hydrogels with various laponite content. Since the laponite clays act as additional cross-linking domains, the swelling ratios (mrel and φ) decrease with increasing laponite content.

Figure 5. The SEM images and EDX analyses of the lyophilized sP(EO-stat-PO) and sP(EO-stat-PO)laponite gels. csP(EO-stat-PO) = 10 wt-% and clap = 1wt-%. Figure 5 shows the inner structures of the lyophilized gels. High resolution SEM analysis revealed that the sP(EO-stat-PO) hydrogel is homogenous, while the nanocomposite gel showed a heterogeneous matrix with embedded clay aggregates. Also, the presence of laponites was confirmed by EDX, of which


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the spectrum showed the peaks for Si (silicon) and Mg (magnesium) (Figure 5). This proves the presence of embedded clays into the matrix while the hydrogel prepared without clays showed no peaks of the respective elements. Functional Scaffolds for Drug Delivery. These nanocomposite hydrogels were uniquely functionalized with BCN motifs for the release of the chemically-bound molecules. The BCN motifs are cyclic functional molecules with a non-benzoannulated cyclooctyne ring, which reacts bioorthogonally with azides through a strain-promoted alkyne-azide reaction (SPAAC), yielding triazole ring. The reaction does not require any metal catalyst, and has been also used in biological milieu without any toxicity. Besides, the triazole ring is resistant to hydrolysis, reduction, oxidation, or other types of cleavage. A wide range of BCN molecules having various functionalities are already available on the marketing shelves, and the reaction rates of these molecules vary in the range of 2 x 10-3 - 1.16 M-1s-1 depending on substituted groups to the cyclooctyne ring, such as electron withdrawing group, fluorine, and azide functional molecule; e.g., BCNs react much faster with electron-deficient aryl azides than aliphatic azides.42


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Figure 6. The hydrogel degradation and the release kinetics of the fluorescence dye. (a) The corresponding photo of the gels under fluorescence light, and (b) the time-dependent release of the dye from the matrix. Filled symbols denote to the release profile from the nanocomposite hydrogel containing BCN-POE3-NH2 molecules (0.22 mmol). clap =1 wt-%. (c) Chemical structure of BCN after clicking with the azide-functional fluorescence dye, and (d) the degradation profile of the nanocomposite hydrogels over time. Figure 6a displays the nanocomposite hydrogels, which were produced with/without azide dye, under fluorescence light. Once the azide dye attached to the scaffold induces fluorescence while the gel without dye displays no fluorescence. The time-dependent release behavior from the hydrogel matrix was monitored with a fluorescence spectrophotometer, and the data are later transformed into the percent (%) release using the control solutions of the dye. Figure 6b shows the initial release of the unbound dyes as first, and followed by the release of covalently bound dyes from the matrix, which ACS Paragon Plus Environment

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occurs slowly, but much faster than the degradation of the hydrogel. One can suppose that the decomposition process of the large triazole moieties triggers fluorescence off, which would more likely deactivate them. However, triazoles are relatively stable rings and do not easily destabilize without certain conditions. Carbamates are chemical groups, which have carbonyl groups linked to oxygen and nitrogen at both sides. There exists more than one bond whose fission can lead to the deprotection of the amine (Figure 6c). Fissions B and C are less probable due to the low reactivity of urethane carbonyl to nucleophiles.43 On the other hand, fission A (alkyl-oxygen fission) is the most probable pathway leading to the deprotection of the urethane-protected amine. The carbamate bond decomposes to the amine liberating CO2 unlike ester cross-linked networks, which are mostly based on lactic or glycolic acids and produce acids upon hydrolysis. The nanocomposite hydrogels were slowly degraded in PBS (Figure 6d). Zhang et al. (2002) reported the degradation of the polymer hydrogel based on lysine diisocyanate (LDI) and glucose.44 The degradation was due to the hydrolysis of carbamate bonds, and varied from couple of weeks to several months depending on the incubation temperature; i.e., the hydrogels degraded at 65% in two months at 37 oC, while the same hydrogel degraded less than 5% at 4 oC. This also supports our finding, which shows less than 6% release of the bound dyes from the gel matrix in 10 days due to gradual network degradation. The Fabrication of Ultrathin Nanocomposite Gel Coatings. One major problem of the nanocomposite systems is their suitability for coating applications, particularly in medical implants. Principally, this is important to benefit from the mechanical and cell binding features of the nanocomposite gels. It is expected that the incorporation of the clays leads to stiffer and mechanically durable nanocomposite gel coatings. In this regard, the presented nanocomposite system can be an ideal solution, since it possesses the in situ network forming property of multifunctional isocyanate endcapped star PEG molecules. On exposure of the NCO-sP(EO-stat-PO)s to an aminofunctional surface, the terminal NCO groups react with surface amines. The residual isocyanates partially hydrolyze into ACS Paragon Plus Environment

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amino groups, and react with the remaining NCO groups so that the ultrathin hydrogel films can be produced. For this reason, the aminosilylated substrates were treated with precursors (NCO-sP(EO-statPO), BCN and laponite), and incubated overnight to ensure complete cross-linking of the components. For the preparation of nanocomposite coatings, water was used as a solvent instead of PBS to avoid possible aggregation of laponite in the coating. After the incubation, the coated-substrates were rinsed with water to remove unbound components, and subsequently dried under a stream of nitrogen.

Figure 7. Characterization of the ultrathin nanocomposite layers. (a) The topological and phase images of the nanocomposite coatings by AFM. (b) Cartoon illustration of the fabrication of ultrathin nanocomposite coatings. (c) The roughness values and contact angles of the coatings. The RMS roughness values (Sq) were calculated over the projected area of 1 µm2. Blank stand for the sample prepared without clays, and the sample "AS" for the aminosilylated surface. (d) XPS spectra of the ACS Paragon Plus Environment

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coatings. Inset shows Mg KLL narrow scan XPS spectra of the coatings. The clay contents in samples 1 and 2 are 2.5 and 5 mg/mL, respectively. The successful fabrication of nanocomposite coating was confirmed by the AFM analyses, which displayed the enhanced surface roughness with increasing content of laponite (Figure 7a, c). Also, the wettability of the coatings was related to the incorporated amount of laponite (Figure 7c). The coating prepared with 0.25 wt-% laponite was more hydrophilic, and had a contact angle of 33o, and the coating prepared with 0.5 wt-% laponite a contact angle of 40o. Higher contact angle could be attributed to higher roughness values (Figure 7c). Layer thicknesses were calculated as 20-25 nm over the AFM topology profiles using Gwyddion Analysis software (ver. 2.44). The quantitative elemental composition of the nanocomposite surface coatings was determined by the XPS analyses (Figure 7d). The nanocomposite layers (samples 1 and 2) showed a significant carbon peak (C) relative to the aminofunctional layer (sample AS). On the other hand, silicone peak (Si) became smaller after the nanocomposite coating because of the sP(EO-stat-PO) molecules. The Mg KLL Auger peak appeared at 305 eV for the nanocomposite coatings, and increased with clay content (Figure 7d, inset). The quantitative elemental composition of the layers was calculated, and the results were presented in Table S1 (see Supporting Information). With increasing amount of laponite incorporation into coatings, C and N contents decreased, and Si and O contents increased, in line with the AFM images (Figure 7a). Biocompatibility of Multifunctional Nanocomposite Gels. An efficient biomaterial for tissue engineering applications should be partially designed on the basis of its influence on the reaction of inflammatory cells, because this is a crucial parameter for biocompatibility.45 Dependent on the behavior of macrophages, polymeric implants have recently been optimized for soft tissue regeneration, specifically blood vessels and heart muscles.46 In addition to material itself, components of the materials should be biocompatible. In this context, isocyanates (NCO) and their bio-applications are important due


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to their toxicity, since other ingredients of the presented system (sP(EO-stat-PO), BCN and laponite) are known as biocompatible.20, 47, 48 Isocyanate (NCO) groups strongly react with amines (NH2) and even with hydroxyl groups (OH) at the physiological pH, and are commonly known to negatively interfere with cellular functions and development.49 However, most of the isocyanates used are hydrophobic molecules with low molecular weight. In contrast, we use PEG-based macromers with terminal NCO groups, which make up less than 3 % of the whole molecule. Additionally, NCO groups hydrolyze into biocompatible amine groups (NH2) in aqueous solutions less than one hour (Figure 8ii). Upon the completion of hydrolysis, the system becomes biocompatible, and there will be no free reactive NCO groups available for uncontrolled reactions.48 This is in line with the results of Landlein and colleagues on the one-step fabrication of multifunctional hydrogels using L-lysine diisocyanate ethyl ester (LDI) with gelatin for bone regeneration where no significant toxicity of the scaffolds induced by diisocyanate linkers in vivo and in vitro was observed.50


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Figure 8. Cellular fluorescence as a reporter of viability. (i) Fluorescent murine macrophages were cultured on different materials to demonstrate viability reporter function of cellular green fluorescence protein signal. Macrophages were cultured for 48 hours on tissue culture treated polystyrol (A-D), on a representative material, a hydrogel consisting of 10% sP(EO-stat-PO) (E-H), and on a toxic hydrogel material (I-L). Propidium iodide staining was performed to determine cell viability. (ii) The mean intensity of NCO peak as a function of time at csP(EO-stat-PO) = 50 wt-% in PBS. (iii) Variation in cell viability in percentage as a function of time for the nanocomposite gels with different laponite concentrations.


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To study cell viability on three-dimensional materials in optical microscopy, we used fluorescent macrophages. The fluorescence of the cells correlated with their viability, meaning that the green fluorescent cells on the control material (Figure 8 A-D) and representative hydrogel (Figure 8 E-H) were viable. In contrast, the dead cells lost their green fluorescence on a toxic material, and exhibited a red signal after propidium iodide staining (Figure 8 I-L). To study the effects of material properties on cell viability, we cultured fluorescent macrophages on these materials. We studied the cell behavior after 2 hours of culture to evaluate the attachment of macrophages on materials coated with the NCO-sP(EOstat-PO) hydrogel, which are known to be cell-repulsive.51 The fluorescence of the cells on the laponitebased materials was very intensive. The initial attachment of macrophages to hydrogels with a laponite content of 0.5% was low, and the cells also exhibited a lower intensity of fluorescence compared to the hydrogel containing 1 wt-% laponite. After 48 hours of culture, macrophages had proliferated on the material (Figure 8i), suggesting the biocompatibility of laponites. Likewise, Gaharwar et al. (2011) showed that increasing laponite content induced higher cell proliferation.52 Given the gelation time, we conclude that the hydrogel having 1 wt-% laponite might in vivo assist in tissue regeneration after injection by raising the cell number, and might assist in healing injured tissues upon injection. In such a case, possible toxicity of free isocyanate groups on cell viability is probably minimized by the laponite clays because of the proliferation of cells (Figure 8iii). CONCLUSION. We present here a novel and versatile nanocomposite system which possesses many desirable gel characteristics, such as injectability, high mechanical strength, degradability, biocompatibility, BCN functionality and suitability for nanolayer coatings. The mechanically strong hydrogels (Ec ~1 MPa) were synthesized by the in situ rapid gelation of the NCO terminal PEG-based macromers in the presence of laponite clays. The network characteristics were examined by several techniques such as rheology, DMA, WAXS, SEM, AFM, TGA, DSC, DLS and contact angle analysis. The cell viability study using murine macrophages showed biocompatibility of the gel system: The cell


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number increased with laponite content. Covalently bounded BCN motifs were used to ligate azide functional molecules, and their gradual release from the matrix was recorded. Furthermore, this gelation system was employed to produce ultrathin hydrogel coatings with thicknesses below 100 nm and tunable wettability together with roughness. With this facile and versatile approach, nanocomposite gels can be either fabricated as bulk hydrogels, or spin-coated to obtain ultrathin films with desired functional motifs. ASSOCIATED CONTENT Results of the rheological, DLS, TGA, DMA and XPS analyses of nanocomposite gels as well as DLS analysis of laponite. This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Corresponding Author * Dr. Fuat Topuz ([email protected]) Author Contributions F. T. designed and conceived the experiments. The experiments were performed by F. T., M. B. and Y. P. All authors have read this final version of the manuscript, and agree with it. ACKNOWLEDGEMENTS F. T. gratefully acknowledges continuous support by Martin Moeller. ABBREVIATIONS sP(EO-stat-PO), star-type poly(ethylene oxide-stat-propylene oxide); NCO, isocyanate; BCN, bicyclononyne. REFERENCES 1.

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