One-Step Fabrication of Biocompatible Multifaceted Nanocomposite

Subscriber access provided by University of Colorado Boulder

Article

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

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.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

One-Step Fabrication of Biocompatible Multifaceted

2

Nanocomposite Gels and Nanolayers

3

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

4

a

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

5

52074 Aachen (Germany)

6

b

7

c

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

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

8 9 10 11

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]

12 13 14 15 16 17 18 19

ACS Paragon Plus Environment

1

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 32

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

shown that the material composition strongly affects cell behavior.

39

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

40 41


2

Page 3 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

42

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

43

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

44

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

45

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

46

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

47

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

48

energy dissipation mechanism in the gel network.5,

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

6

In addition to the permanent cross-links, the

61

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

62

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

63

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

64

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

65

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


3

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 32

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

gel networks.22, 23

77 78

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

79

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


4

Page 5 of 32

1 2 80 3 4 81 5 6 7 8 82 9 10 83 11 12 84 13 14 15 85 16 17 86 18 19 87 20 21 22 88 23 24 89 25 26 90 27 28 29 91 30 31 92 32 33 34 93 35 36 94 37 38 95 39 40 41 42 96 43 44 97 45 46 98 47 48 49 99 50 51100 52 53 101 54 55 56102 57 58103 59 60

Biomacromolecules

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

5

Biomacromolecules

1 2 104 3 4 105 5 6 7 106 8 9 107 10 11108 12 13 14109 15 16110 17 18 111 19 20 21112 22 23113 24 25 114 26 27 28115 29 30116 31 32 33117 34 35118 36 37119 38 39 40120 41 42121 43 44 45122 46 47123 48 49 124 50 51 52125 53 54126 55 56 57127 58 59 60

Page 6 of 32

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.


6

Page 7 of 32

1 2 128 3 4 129 5 6 7 130 8 9 131 10 11 12132 13 14133 15 16134 17 18 19135 20 21136 22 23 137 24 25 26138 27 28139 29 30 31140 32 33141 34 35142 36 37 38143 39 40144 41 42 145 43 44 45146 46 47147 48 49 148 50 51 52149 53 54150 55 56 57151 58 59152 60

Biomacromolecules

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

(1)

mrel= (m/mo)

(2)

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

7

Biomacromolecules

1 2 153 3 4 154 5 6 7 155 8 9 156 10 11 157 12 13 14158 15 16159 17 18 19160 20 21161 22 23162 24 25 26163 27 28164 29 30 31165 32 33166 34 35 167 36 37 38168 39 40169 41 42 43 170 44 45 46171 47 48172 49 50 173 51 52 53174 54 55175 56 57 58 59 60

Page 8 of 32

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.


8

Page 9 of 32

1 2 176 3 4 177 5 6 7 178 8 9 179 10 11180 12 13 14181 15 16182 17 18 183 19 20 21184 22 23185 24 25 186 26 27 28187 29 30188 31 32 33189 34 35190 36 37191 38 39 40192 41 42193 43 44 194 45 46 47195 48 49196 50 51 52197 53 54198 55 56199 57 58 59 60

Biomacromolecules

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


9

Biomacromolecules

1 2 200 3 4 201 5 6 7 202 8 9 203 10 11204 12 13 14205 15 16 17206 18 19207 20 21 22208 23 24209 25 26210 27 28 29211 30 31212 32 33 213 34 35 36214 37 38215 39 40 41216 42 43217 44 45218 46 47 48219 49 50220 51 52 221 53 54 55222 56 57223 58 59 60

Page 10 of 32

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,


10

Page 11 of 32

1 2 224 3 4 225 5 6 7 226 8 9 227 10 11228 12 13 14229 15 16230 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44231 45 46 47232 48 49233 50 51234 52 53 54235 55 56236 57 58 237 59 60

Biomacromolecules

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

11

Biomacromolecules

1 2 238 3 4 239 5 6 7 240 8 9 10241 11 12242 13 14 15243 16 17244 18 19 245 20 21 22246 23 24247 25 26 27248 28 29249 30 31 250 32 33 34251 35 36252 37 38 39253 40 41254 42 43255 44 45 46256 47 48257 49 50 258 51 52 53259 54 55260 56 57 58 59 60

Page 12 of 32

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


12

Page 13 of 32

1 2 261 3 4 262 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36263 37 38264 39 40265 41 42 266 43 44 45267 46 47268 48 49 50269 51 52270 53 54271 55 56 57 58 59 60

Biomacromolecules

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.


13

Biomacromolecules

1 2 272 3 4 273 5 6 7 274 8 9 275 10 11276 12 13 14277 15 16278 17 18 279 19 20 21280 22 23281 24 25 282 26 27 28283 29 30284 31 32 33285 34 35286 36 37287 38 39 40288 41 42289 43 44 290 45 46 47291 48 49292 50 51 52293 53 54294 55 56295 57 58 59296 60

Page 14 of 32

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

14

Page 15 of 32

1 2 297 3 4 298 5 6 7 299 8 9 300 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34301 35 36302 37 38 39303 40 41304 42 43305 44 45 46306 47 48307 49 50 51 52308 53 54309 55 56310 57 58 59311 60

Biomacromolecules

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

15

Biomacromolecules

1 2 312 3 4 313 5 6 7 314 8 9 315 10 11316 12 13 14317 15 16318 17 18 19319 20 21320 22 23 24321 25 26322 27 28 323 29 30 31324 32 33325 34 35 36326 37 38327 39 40328 41 42 43329 44 45330 46 47 331 48 49 50332 51 52333 53 54 55334 56 57335 58 59 60

Page 16 of 32

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


16

Page 17 of 32

1 2 336 3 4 337 5 6 7 338 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44339 45 46 47340 48 49 341 50 51 52 53342 54 55343 56 57 344 58 59 60

Biomacromolecules

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


17

Biomacromolecules

1 2 345 3 4 346 5 6 7 347 8 9 10348 11 12349 13 14 15350 16 17351 18 19352 20 21 22353 23 24354 25 26355 27 28 29356 30 31357 32 33 358 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 32

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


18

Page 19 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29359 30 31 360 32 33 34361 35 36362 37 38 363 39 40 41364 42 43365 44 45 46 47366 48 49367 50 51368 52 53 54369 55 56370 57 58 371 59 60

Biomacromolecules

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

19

Biomacromolecules

1 2 372 3 4 373 5 6 7 374 8 9 375 10 11376 12 13 14377 15 16378 17 18 379 19 20 21380 22 23381 24 25 382 26 27 28383 29 30384 31 32 33385 34 35386 36 37387 38 39 40388 41 42389 43 44 390 45 46 47391 48 49392 50 51 52393 53 54394 55 56395 57 58 59396 60

Page 20 of 32

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

20

Page 21 of 32

1 2 397 3 4 398 5 6 7 399 8 9 400 10 11401 12 13 14402 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47403 48 49404 50 51 405 52 53 54406 55 56407 57 58 59408 60

Biomacromolecules

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

21

Biomacromolecules

1 2 409 3 4 410 5 6 7 8 411 9 10412 11 12413 13 14 15414 16 17415 18 19 416 20 21 22417 23 24418 25 26 419 27 28 29420 30 31421 32 33 34422 35 36423 37 38424 39 40 41425 42 43426 44 45 427 46 47 48428 49 50429 51 52 430 53 54 55431 56 57 58 59 60

Page 22 of 32

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


22

Page 23 of 32

1 2 432 3 4 433 5 6 7 434 8 9 435 10 11436 12 13 14437 15 16438 17 18 439 19 20 21440 22 23441 24 25 442 26 27 28443 29 30444 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

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


23

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39445 40 41446 42 43 44447 45 46448 47 48449 49 50 51450 52 53451 54 55452 56 57 58453 59 60

Page 24 of 32

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.


24

Page 25 of 32

1 2 454 3 4 455 5 6 7 456 8 9 457 10 11458 12 13 14459 15 16460 17 18 461 19 20 21462 22 23463 24 25 464 26 27 28465 29 30466 31 32 33467 34 35468 36 37469 38 39 40470 41 42 43471 44 45472 46 47 48473 49 50474 51 52 475 53 54 55476 56 57477 58 59 60

Biomacromolecules

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


25

Biomacromolecules

1 2 478 3 4 479 5 6 7 480 8 9 481 10 11482 12 13 14483 15 16 17484 18 19 485 20 21 22486 23 24 25487 26 27 28488 29 30 31489 32 33 490 34 35 36491 37 38 492 39 40 41 42493 43 44494 45 46 47495 48 49496 50 51 52497 53 54 55498 56 57499 58500 59 60

Page 26 of 32

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.

Topuz, F.; Buenger, D.; Tanaka, D.; Groll, J., 3.329 - Hydrogels in Biosensing Applications A2 Ducheyne, Paul. In Comprehensive Biomaterials, Elsevier: Oxford, 2011; pp 491-517. ACS Paragon Plus Environment

26

Page 27 of 32

1 2 501 3 502 4 5 6 503 7 504 8 9 505 10 11506 12 13507 14 508 15 16 17509 18510 19 20511 21512 22 23513 24 25514 26515 27 28 516 29 30517 31 32518 33 34519 35 36520 37521 38 39522 40 41523 42 524 43 44525 45 46526 47 527 48 49528 50 51529 52 53530 54 55531 56532 57 58 59 60

Biomacromolecules

2.

Buenger, D.; Topuz, F.; Groll, J., Hydrogels in sensing applications. Progress in Polymer Science 2012, 37, (12), 1678-1719.

3.

Li, Y.; Rodrigues, J.; Tomas, H., Injectable and biodegradable hydrogels: gelation, biodegradation and biomedical applications. Chemical Society Reviews 2012, 41, (6), 2193-2221.

4.

Vermonden, T.; Censi, R.; Hennink, W. E., Hydrogels for Protein Delivery. Chemical Reviews 2012, 112, (5), 2853-2888.

5.

Seliktar, D., Designing Cell-Compatible Hydrogels for Biomedical Applications. Science 2012, 336, (6085), 1124-1128.

6.

Sharifi, S.; Blanquer, S. B. G.; van Kooten, T. G.; Grijpma, D. W., Biodegradable nanocomposite hydrogel structures with enhanced mechanical properties prepared by photocrosslinking solutions of poly(trimethylene carbonate)–poly(ethylene glycol)–poly(trimethylene carbonate) macromonomers and nanoclay particles. Acta Biomaterialia 2012, 8, (12), 4233-4243.

7.

Deng, G.; Tang, C.; Li, F.; Jiang, H.; Chen, Y., Covalent Cross-Linked Polymer Gels with Reversible Sol−Gel Transition and Self-Healing Properties. Macromolecules 2010, 43, (3), 11911194.

8.

Cordier, P.; Tournilhac, F.; Soulie-Ziakovic, C.; Leibler, L., Self-healing and thermoreversible rubber from supramolecular assembly. Nature 2008, 451, (7181), 977-980.

9.

Tuncaboylu, D. C.; Sari, M.; Oppermann, W.; Okay, O., Tough and Self-Healing Hydrogels Formed via Hydrophobic Interactions. Macromolecules 2011, 44, (12), 4997-5005.

10.

Liu, J.; Chen, C.; He, C.; Zhao, J.; Yang, X.; Wang, H., Synthesis of Graphene Peroxide and Its Application in Fabricating Super Extensible and Highly Resilient Nanocomposite Hydrogels. ACS Nano 2012, 6, (9), 8194-8202.

11.

Banik, S. J.; Fernandes, N. J.; Thomas, P. C.; Raghavan, S. R., A New Approach for Creating Polymer Hydrogels with Regions of Distinct Chemical, Mechanical, and Optical Properties. Macromolecules 2012, 45, (14), 5712-5717.

12.

Dalton, P. D.; Hostert, C.; Albrecht, K.; Moeller, M.; Groll, J., Structure and Properties of UreaCrosslinked Star Poly[(ethylene oxide)-ran-(propylene oxide)] Hydrogels. Macromolecular Bioscience 2008, 8, (10), 923-931.

13.

Na, Y. H., Double network hydrogels with extremely high toughness and their applications. Korea-Australia Rheology Journal 2013, 25, (4), 185-196.

14.

Hao, J.; Weiss, R. A., Viscoelastic and Mechanical Behavior of Hydrophobically Modified Hydrogels. Macromolecules 2011, 44, (23), 9390-9398.


27

Biomacromolecules

1 2 533 3 534 4 5 535 6 7 536 8 537 9 10538 11 12539 13 540 14 15541 16542 17 18 19543 20544 21 22545 23 24546 25547 26 27 548 28 29549 30550 31 551 32 33 34552 35553 36 37554 38 39555 40556 41 42557 43558 44 45 559 46 47560 48561 49 50 51562 52563 53 54564 55 56565 57566 58 59 60

Page 28 of 32

15.

Karpovich, A. L.; Vlasova, M. F.; Sapronova, N. I.; Sukharev, V. S.; Ivanov, V. V., Determination of dimensions of exfoliating materials in aqueous suspensions. MethodsX 2016, 3, 19-24.

16.

Negrete-Herrera, N.; Putaux, J.-L.; Bourgeat-Lami, E., Synthesis of polymer/Laponite nanocomposite latex particles via emulsion polymerization using silylated and cation-exchanged Laponite clay platelets. Progress in Solid State Chemistry 2006, 34, (2-4), 121-137.

17.

Gaharwar, A. K.; Kishore, V.; Rivera, C.; Bullock, W.; Wu, C.-J.; Akkus, O.; Schmidt, G., Physically Crosslinked Nanocomposites from Silicate-Crosslinked PEO: Mechanical Properties and Osteogenic Differentiation of Human Mesenchymal Stem Cells. Macromolecular Bioscience 2012, 12, (6), 779-793.

18.

Jugdaohsingh, R., Silicone and bone health. The journal of nutrition, health & aging 2007, 11, (2), 99-110.

19.

Jurkić, L. M.; Cepanec, I.; Pavelić, S. K.; Pavelić, K., Biological and therapeutic effects of orthosilicic acid and some ortho-silicic acid-releasing compounds: New perspectives for therapy. Nutrition & Metabolism 2013, 10, (1), 2.

20.

Gaharwar, A. K.; Mihaila, S. M.; Swami, A.; Patel, A.; Sant, S.; Reis, R. L.; Marques, A. P.; Gomes, M. E.; Khademhosseini, A., Bioactive Silicate Nanoplatelets for Osteogenic Differentiation of Human Mesenchymal Stem Cells. Advanced Materials 2013, 25, (24), 33293336.

21.

Gaharwar, A. K.; Avery, R. K.; Assmann, A.; Paul, A.; McKinley, G. H.; Khademhosseini, A.; Olsen, B. D., Shear-Thinning Nanocomposite Hydrogels for the Treatment of Hemorrhage. ACS Nano 2014, 8, (10), 9833-9842.

22.

Tamesue, S.; Ohtani, M.; Yamada, K.; Ishida, Y.; Spruell, J. M.; Lynd, N. A.; Hawker, C. J.; Aida, T., Linear versus Dendritic Molecular Binders for Hydrogel Network Formation with Clay Nanosheets: Studies with ABA Triblock Copolyethers Carrying Guanidinium Ion Pendants. Journal of the American Chemical Society 2013, 135, (41), 15650-15655.

23.

Wang, Q.; Mynar, J. L.; Yoshida, M.; Lee, E.; Lee, M.; Okuro, K.; Kinbara, K.; Aida, T., Highwater-content mouldable hydrogels by mixing clay and a dendritic molecular binder. Nature 2010, 463, (7279), 339-343.

24.

Lee, S.; Kim, H. J.; Chang, S. H.; Lee, J., Anisometric nanocomposite hydrogels with temperature responsive compartments. Soft Matter 2013, 9, (2), 472-479.

25.

Wang, M.; Yuan, D.; Fan, X.; Sahoo, N. G.; He, C., Polymer Nanocomposite Hydrogels Exhibiting Both Dynamic Restructuring and Unusual Adhesive Properties. Langmuir 2013, 29, (23), 7087-7095.


28

Page 29 of 32

1 2 567 3 568 4 5 569 6 570 7 8 571 9 10572 11 12573 13 574 14 15 16575 17576 18 19577 20 21578 22579 23 24580 25 26581 27 582 28 29583 30 31584 32 585 33 34586 35 36587 37 38588 39589 40 41590 42 43591 44592 45 46 593 47 48594 49595 50 51 52596 53597 54598 55 56 57 58 59 60

Biomacromolecules

26.

Kochumalayil, J. J.; Morimune, S.; Nishino, T.; Ikkala, O.; Walther, A.; Berglund, L. A., NacreMimetic Clay/Xyloglucan Bionanocomposites: A Chemical Modification Route for Hygromechanical Performance at High Humidity. Biomacromolecules 2013, 14, (11), 38423849.

27.

Gaharwar, A. K.; Peppas, N. A.; Khademhosseini, A., Nanocomposite hydrogels for biomedical applications. Biotechnology and Bioengineering 2014, 111, (3), 441-453.

28.

Baumann, M. D.; Kang, C. E.; Tator, C. H.; Shoichet, M. S., Intrathecal delivery of a polymeric nanocomposite hydrogel after spinal cord injury. Biomaterials 2010, 31, (30), 7631-7639.

29.

Appel, E. A.; Tibbitt, M. W.; Webber, M. J.; Mattix, B. A.; Veiseh, O.; Langer, R., Selfassembled hydrogels utilizing polymer-nanoparticle interactions. Nature Communications 2015, 6, 9, 6295.

30.

Kim, Y. S.; Liu, M.; Ishida, Y.; Ebina, Y.; Osada, M.; Sasaki, T.; Hikima, T.; Takata, M.; Aida, T., Thermoresponsive actuation enabled by permittivity switching in an electrostatically anisotropic hydrogel. Nat Mater 2015, 14, (10), 1002-1007.

31.

Liu, M. J.; Ishida, Y.; Ebina, Y.; Sasaki, T.; Aida, T., Photolatently modulable hydrogels using unilamellar titania nanosheets as photocatalytic crosslinkers. Nature Communications 2013, 4, 2029.

32.

Haraguchi, K.; Murata, K.; Takehisa, T., Stimuli-Responsive Nanocomposite Gels and Soft Nanocomposites Consisting of Inorganic Clays and Copolymers with Different Chemical Affinities. Macromolecules 2012, 45, (1), 385-391.

33.

Annaka, M.; Mortensen, K.; Matsuura, T.; Ito, M.; Nochioka, K.; Ogata, N., Organic-inorganic nanocomposite gels as an in situ gelation biomaterial for injectable accommodative intraocular lens. Soft Matter 2012, 8, (27), 7185-7196.

34.

García-Astrain, C.; Chen, C.; Burón, M.; Palomares, T.; Eceiza, A.; Fruk, L.; Corcuera, M. Á.; Gabilondo, N., Biocompatible Hydrogel Nanocomposite with Covalently Embedded Silver Nanoparticles. Biomacromolecules 2015, 16, (4), 1301-1310.

35.

Merino, S.; Martín, C.; Kostarelos, K.; Prato, M.; Vázquez, E., Nanocomposite Hydrogels: 3D Polymer-Nanoparticle Synergies for On-Demand Drug Delivery. ACS Nano 2015, 9, (5), 46864697.

36.

Yang, J.; Liu, S.; Xiao, Y.; Gao, G.; Sun, Y.; Guo, Q.; Wu, J.; Fu, J., Multi-responsive nanocomposite hydrogels with high strength and toughness. Journal of Materials Chemistry B 2016, 4, (9), 1733-1739.


29

Biomacromolecules

1 2 599 3 600 4 5 601 6 7 602 8 603 9 10604 11 12605 13 606 14 15 16607 17608 18 19 20609 21610 22 23611 24 25612 26613 27 614 28 29 30615 31616 32 33 34617 35618 36619 37 38 39620 40621 41 42 622 43 44623 45624 46 625 47 48 49626 50627 51 52628 53629 54 55630 56 57631 58632 59 60

Page 30 of 32

37.

Götz, H.; Beginn, U.; Bartelink, C. F.; Grünbauer, H. J. M.; Möller, M., Preparation of Isophorone Diisocyanate Terminated Star Polyethers. Macromolecular Materials and Engineering 2002, 287, (4), 223-230.

38.

Nie, J.; Du, B.; Oppermann, W., Swelling, Elasticity, and Spatial Inhomogeneity of Poly(Nisopropylacrylamide)/Clay Nanocomposite Hydrogels. Macromolecules 2005, 38, (13), 57295736.

39.

Okay, O.; Oppermann, W., Polyacrylamide−Clay Nanocomposite Hydrogels: Rheological and Light Scattering Characterization. Macromolecules 2007, 40, (9), 3378-3387.

40.

Li, J.; Illeperuma, W. R. K.; Suo, Z.; Vlassak, J. J., Hybrid Hydrogels with Extremely High Stiffness and Toughness. ACS Macro Letters 2014, 3, (6), 520-523.

41.

Lake, G. J.; Thomas, A. G., The Strength of Highly Elastic Materials. Proceedings of the Royal Society of London. Series A. Mathematical and Physical Sciences 1967, 300, (1460), 108-119.

42.

Dommerholt, J.; Schmidt, S.; Temming, R.; Hendriks, L. J. A.; Rutjes, F. P. J. T.; van Hest, J. C. M.; Lefeber, D. J.; Friedl, P.; van Delft, F. L., Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells. Angewandte Chemie International Edition 2010, 49, (49), 9422-9425.

43.

Sureshbabu, V. V.; Narendra, N., Protection Reactions. In Amino Acids, Peptides and Proteins in Organic Chemistry, Wiley-VCH Verlag GmbH & Co. KGaA: 2011; pp 1-97.

44.

Zhang, J. Y.; Beckman, E. J.; Hu, J.; Yang, G. G.; Agarwal, S.; Hollinger, J. O., Synthesis, biodegradability, and biocompatibility of lysine diisocyanate-glucose polymers. Tissue Engineering 2002, 8, (5), 771-785.

45.

Remes, A.; Williams, D. F., Immune response in biocompatibility. Biomaterials 1992, 13, (11), 731-743.

46.

Zachman, A. L.; Crowder, S. W.; Ortiz, O.; Zienkiewicz, K. J.; Bronikowski, C. M.; Yu, S. S.; Giorgio, T. D.; Guelcher, S. A.; Kohn, J.; Sung, H.-J., Pro-angiogenic and Anti-inflammatory Regulation by Functional Peptides Loaded in Polymeric Implants for Soft Tissue Regeneration. Tissue Engineering. Part A 2013, 19, (3-4), 437-447.

47.

Lang, K.; Davis, L.; Wallace, S.; Mahesh, M.; Cox, D. J.; Blackman, M. L.; Fox, J. M.; Chin, J. W., Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels–Alder Reactions. Journal of the American Chemical Society 2012, 134, (25), 10317-10320.

48.

Groll, J.; Fiedler, J.; Engelhard, E.; Ameringer, T.; Tugulu, S.; Klok, H.-A.; Brenner, R. E.; Moeller, M., A novel star PEG–derived surface coating for specific cell adhesion. Journal of Biomedical Materials Research Part A 2005, 74A, (4), 607-617. ACS Paragon Plus Environment

30

Page 31 of 32

1 2 633 3 634 4 5 635 6 7 636 8 637 9 10638 11639 12 13 640 14 641 15 16642 17 18 19643 20644 21645 22 23 24646 25 26647 27 28 648 29 30 31649 32 33650 34 35 36651 37 38652 39 40 653 41 42 43654 44 45655 46 47 48656 49 50657 51 52658 53 54 55659 56 57660 58 59661 60

Biomacromolecules

49.

Raghuram, G. V.; Pathak, N.; Jain, D.; Panwar, H.; Pandey, H.; Jain, S. K.; Mishra, P. K., Molecular mechanisms of isocyanate induced oncogenic transformation in ovarian epithelial cells. Reproductive Toxicology 2010, 30, (3), 377-386.

50.

Neffe, A. T.; Pierce, B. F.; Tronci, G.; Ma, N.; Pittermann, E.; Gebauer, T.; Frank, O.; Schossig, M.; Xu, X.; Willie, B. M.; Forner, M.; Ellinghaus, A.; Lienau, J.; Duda, G. N.; Lendlein, A., One Step Creation of Multifunctional 3D Architectured Hydrogels Inducing Bone Regeneration. Advanced Materials 2015, 27, (10), 1738-1744.

51.

Bartneck, M.; Heffels, K.-H.; Pan, Y.; Bovi, M.; Zwadlo-Klarwasser, G.; Groll, J., Inducing healing-like human primary macrophage phenotypes by 3D hydrogel coated nanofibres. Biomaterials 2012, 33, (16), 4136-4146.

52.

Gaharwar, A. K.; Schexnailder, P. J.; Kline, B. P.; Schmidt, G., Assessment of using Laponite® cross-linked poly(ethylene oxide) for controlled cell adhesion and mineralization. Acta Biomaterialia 2011, 7, (2), 568-577.


31

Biomacromolecules

1 2 662 3 4 663 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21664 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 32

Table of Contents (ToC) Graphic


32

One-Step Fabrication of Biocompatible Multifaceted Nanocomposite

Recommend Documents