Caddisfly Inspired Phosphorylated Poly(ester urea)-Based

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Caddisfly Inspired Phosphorylated Poly(esterurea) based Degradable Bone Adhesives Vrushali Bhagat, Emily O'Brien, Jinjun Zhou, and Matthew L. Becker Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00875 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 17, 2016

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Caddisfly Inspired Phosphorylated Poly(ester-urea)

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based Degradable Bone Adhesives

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Vrushali Bhagat, Emily O’Brien, Jinjun Zhou† and Matthew L. Becker* Department of Polymer Science, The University of Akron, Akron, OH-44325, USA

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ABSTRACT

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Bone and tissue adhesives are essential in surgeries for wound healing, hemostasis, tissue

8

reconstruction and drug delivery. However, there are very few degradable materials with high

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adhesion strengths that degrade into bioresorbable byproducts. Caddisfly adhesive silk is

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interesting due to the presence of phosphoserines which are thought to afford adhesive

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properties. In this work phosphoserine-valine poly(ester urea) copolymers with 2% and 5%

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phosphoserine content were synthesized to mimic caddisfly adhesive silk. Significantly the

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materials are ethanol soluble and water insoluble making them clinically relevant. Their physical

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properties were quantified and the adhesion properties were studied on aluminum and bovine

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bone substrates before and after crosslinking with Ca2+ ions. The adhesive strength of the

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phosphorylated copolymer on a bone substrate after crosslinking with Ca2+ was 439 ± 203 KPa,

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comparable to commercially available PMMA bone cement (530 ± 133 KPa).

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KEYWORDS: Caddisfly, poly(ester-urea), phosphoserine, tissue adhesives, adhesion strength

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ACS Paragon Plus Environment

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INTRODUCTION

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Bone and tissue adhesives are essential in surgeries for their assistance in wound healing,

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hemostasis, tissue reconstruction and drug delivery.1 Classical structural alternatives in bone

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surgeries include use of metallic plates, pins and screws as support medium. These options while

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safe and serviceable suffer from aseptic loosening, poor anchorage to the bone, cause irritation to

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the neighboring soft tissue, cause discomfort to the patient and need to be replaced or removed

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after the bone regeneration. Poly(methyl methacrylate) (PMMA) bone cement is also commonly

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used in bone surgeries however, it is not adhesive in nature, lack chemical interaction, cause

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significant heat generation and shrinkage. Attempts to use synthetic glues like cyanoacrylate,

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polyurethane, epoxy resin and calcium or magnesium phosphate ceramic bone cement as bone

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adhesives have failed either due to lack of interaction with the bone surface or poor strength.2

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Alternative, bone adhesives with degradable properties and high adhesion strengths are currently

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an attractive clinical target.

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Phosphate based compounds have been used as adhesion promoters for decades in underwater

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coatings, dental applications, bone implants, fillers and metal substrates.3-8 Several studies have

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demonstrated adhesion or bonding strength and osteoconductive potential of phosphate

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functionalized polymeric bone grafts showing significant improvement in bone bonding.5,

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Recently Breucker et.al. developed a facile route to phosphorus functionalized polyurethane

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aqueous dispersions. Quartz crystal microbalance (QCM) studies showed enhanced affinity of

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the dispersions towards hydroxyapatite and stainless steel surfaces suggesting their application as

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potential bone adhesives.18 Clearly, phosphate containing polypeptides or polymers demonstrate

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improved adhesion behavior on hydroxyapatite or bone surface and show promise for next

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generation bone adhesives.

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Marine organisms like mussels, barnacles, starfish, sanscastle worms and caddisflies have

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evolved to synthesize their own underwater adhesive with strong bonding characteristics.19

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Studies on caddisfly adhesive silk have confirmed the presence of phosphoserine (pSer) residues

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in the form of (SX)4 motifs where S is serine and X is usually valine or isoleucine. Elemental

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analysis also showed the presence of divalent cations like Ca2+ and Mg2+ which undergo strong

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electrostatic interaction with the phosphate groups in pSer to impart strength to the fibers. The

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adhesive nature of caddisflies is attributed to the heavily phosphorylated regions in the adhesive

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filament.20-24 Recent studies by Wang et.al. describe an alternative model of caddisfly silk

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adhesion consisting of a crosslinked dityrosine peripheral layer and an evenly distributed PEVK

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like protein throughout the fiber core. The dityrosines crosslinked in the presence of reactive

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oxygen species were thought to impart strength and adhesion to the fibers.25, 26 However, the role

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of phosphate groups as adhesion promoters in caddisfly silk was not completely ruled out. In this

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work, inspired from the caddisfly adhesive silk, a phosphate functionalized poly(ester urea) was

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created as a bone adhesive.

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Poly(ester urea)s (PEU)s are a class of polymers well suited for biomaterial applications

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because of their attractive properties including degradation into metabolic components, tunable

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mechanical properties, wide range of functionality and nontoxicity in vitro and in vivo.27-31 PEU

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copolymers based on pSer and valine amino acid, as found in caddisfly silk were synthesized and

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characterized foe their physical properties. The pSer content incorporated in the copolymer was

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2% and 5%. The solubility of these polymers in ethanol makes them more clinically relevant and

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show strong possibility for further development as bone adhesives. Their adhesion strengths

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were studied before and after crosslinking with Ca2+ by lap shear adhesion on aluminum

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substrates and end-to-end adhesion on bovine bone substrates. 3


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EXPERIMENTAL SECTION

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Materials and Methods. L-valine, p-toluene sulfonic acid monohydrate, toluene, 1,8-octanediol,

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anhydrous dichloromethane (DCM), Pd/C, PtO2, ethanol, diphenylphosphoryl chloride (99%),

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anhydrous pyridine, 4M HCl/Dioxane solution, chloroform, trimethylamine and acetic acid were

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purchased from Sigma Aldrich. N-Boc-O-Bzl-L-Serine was purchased from Ark Pharm, Inc.

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N,N-Diisopropyl carbodiimide (DIC) was purchased from Oakwood Chemicals. Triphosgene

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was purchased from TCI America. 4-(dimethyl amino) pyridinium 4-toluenesulfonate (DPTS)

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was synthesized according to literature procedure. Chloroform was dried over CaH2 overnight,

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distilled and stored in dark before use. All chemicals were used as received unless noted

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otherwise.

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1

H,

13

C and

31

P NMR spectra of monomers and polymers were recorded on a Varian NMR

78

Spectrophotometer (300 MHz and 500 MHz respectively). Chemical shifts (δ) were reported in

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ppm and referenced to residual solvent resonances (1H NMR, DMSO-d6: δ = 2.50 ppm and 13C

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NMR, DMSO-d6: δ = 39.50 ppm). Abbreviations of multiplicities are denoted as s-singlet, d-

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doublet, m-multiplet, q-quartet, dd-double doublet, td-triple doublet. Attenuated Total Internal

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Reflection Infrared (ATR-IR) Spectra of polymers were recorded on Shimadzu Miracle 10 ATR-

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FTIR equipped with a quartz crystal window. A small piece of polymer, enough to cover the

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crystal window was used for the measurements. Electrospray Ionization Mass spectrum (ESI-

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MS) of monomer was recorded on Bruker HCTultra II quadrupole ion trap (QIT) mass

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spectrometer (Billerica, MA) equipped with an ESI source. Molecular weights and PDI (ÐM) of

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the polymers were determined by Size exclusion chromatography on TOSOH ECOSEC HLC4


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8320GPC using DMF (0.1 M LiBr salt solution) as eluent at a flow rate of 0.5 mL/min at 50 °C

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equipped with a refractive index detector. The glass transition temperature (Tg) of polymers was

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determined by Differential Scanning Calorimetry (DSC, TA Q200) at a scanning rate of 10

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°

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determined using Thermogravimetric Analysis (TGA, TA Q500) at a heating rate of 10 °C/min

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from 25 °C to 600 °C.

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C/min for 5 cycles from -50 °C to 60 °C. The decomposition temperatures of polymers (Td) were

The synthesis and characterization of all the monomers is described in detail in the supporting

95

information.

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Synthesis

of

1,8-octanediol-L-valine

poly(ester

urea)

(Poly(1-Val-8)):

A

solution

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polymerization was carried out by dissolving the di-p-toluenesulfonic acid salt of bis(L-Valine)-

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1,8-Octanyl-diester (M1) (13.74 g, 20 mmol, 1 eq.) and triethylamine (12.55 ml, 90 mmol, 4.5

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eq.) in 40 mL of chloroform in a 500 mL three neck round bottom flask equipped with a

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magnetic stir bar and a pressure equalizing addition funnel. Triphosgene (2.37 g, 8 mmol, 0.4

101

eq.) was then dissolved in 15 mL of freshly distilled chloroform and added dropwise under

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nitrogen at room temperature. The reaction continued for 12 h after which an additional aliquot

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of triphosgene (0.45 g, 1.5 mmol, 0.08 eq.) dissolved in 15 mL of chloroform was added to the

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flask dropwise. The reaction was stirred for an additional 8 h. The polymer was purified by

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precipitation in hot water and dried under vacuum. 1H NMR (500 MHz, DMSO-d6, δ): 0.86 (m,

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12H, CH3), 1.26 (s, 8H, CH2), 1.54 (s, 4H, CH2), 1.98 (dd, J = 6.69 Hz, 11.73 Hz, 2H; CH),

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3.78-4.21 (m, 6H, CH2, CH), 6.37 (d, J = 8.89 Hz, 2H; NH). 13C NMR (500 MHz, DMSO-d6, δ):

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19.39 (C4), 25.69 (C2), 28.55 (C2), 28.97 (C2), 30.91 (C2), 58.13 (C2), 64.52 (C2), 157.96 (NH-

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C=O), 172.84 (O-C=O). ATR-IR (ν): 1650-1690 cm-1 (s, C=O urea stretch), 1735-1750 cm-1 (s,

5


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C=O ester stretch), 3300-3500 cm-1 (m, N-H urea stretch). ÐM = 3.2, Mn = 4.3 KDa, Mw = 13.9

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KDa.

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Synthesis

of

Poly(serine

diphenylphosphate-co-valine)

(Poly(SerDPP-co-Val):

The

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copolymer was synthesized via a solution polymerization similar to P(1-Val-8). Typically, both

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the monomers (20 mmol, 1 eq.) and triethylamine (90 mmol, 4.5 eq.) were dissolved in 40 mL of

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freshly distilled CHCl3 under N2 in a 500 mL three neck round bottom flask equipped with a

116

magnetic stirrer. Triphosgene (8 mmol, 0.4 eq.) was dissolved in 15 mL of distilled chloroform

117

and added dropwise to the flask under nitrogen at room temperature. The reaction continued for

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12 h and then additional aliquot of triphosgene (1.5 mmol, 0.075 eq in 10 mL of chloroform) was

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added dropwise to the flask. The reaction continued for another 8 h after which the reaction

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solution was transferred to a separatory funnel and precipitated dropwise in hot water. After the

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water cools to ambient temperature, the polymer was washed in water at room temperature

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overnight and finally dried under vacuum to obtain white polymer as product.

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5% Poly(SerDPP-co-Val): Theoretical monomer feed ratio- pSer/Val (M5/M1) = 15/85: 1H

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NMR (500 MHz, DMSO-d6, δ): Actual Monomer ratio = 5/95, δ = 0.85 (dd, J = 6.76 Hz, 13.63

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Hz, 195H; CH3), 1.26 (d, J = 11.12 Hz, 154H; CH2), 1.54 (m, 72H, CH2), 1.98 (dd, J = 6.56 Hz,

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12.58 Hz, 31H; CH), 3.93-4.14 (m, 137H, CH2, CH), 6.37 (d, J = 8.91 Hz, 38H; NH), 7.06-7.18

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(m, 26H, Ar H), 7.19-7.34 (m, 12H, Ar H). 13C NMR (500 MHz, DMSO-d6, δ): 19.4 (C4), 25.69

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(C4), 28.55 (C4), 30.90 (C2), 58.15 (C4), 64.53 (C4), 120.34 (Ar C), 123.08 (Ar C), 129.31 (Ar

129

C), 157.97 (NH-C=O), 172.85 (O-C=O).

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reference, δ): 11.58 ppm. ATR-IR (ν): 1650-1690 cm-1 (s, C=O urea stretch), 1735-1750 cm-1 (s,

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C=O ester stretch), 3300-3500 cm-1 (m, N-H urea stretch), ~1040 cm-1 (w, P=O stretch), ~675

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cm-1 (w, P-O stretch). ÐM = 1.4, Mn = 7.7 KDa, Mw = 10.5 KDa.

31

P NMR (500 MHz, DMSO-d6, 85% H3PO4 external

6


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2% Poly(SerDPP-co-Val): Theoretical monomer feed ratio- pSer/Val (M5/M1) = 10/90: 1H

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NMR spectra resonances are similar to 5% P(SerDPP-co-Val) with different integration values.

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Actual Monomer ratio = 2/98. 13C and 31P NMR spectra and ATR-IR spectra corresponds with

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that of 5% Poly(SerDPP-co-Val). ÐM = 1.4, Mn = 6.5 KDa, Mw = 9.3 KDa.

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Synthesis of Poly(phospho serine-co-valine) (Poly(pSer-co-Val): The diphenyl protecting

138

groups were deprotected by hydrogenolysis in the presence of PtO2 catalyst to synthesize

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phosphate functionalized copolymers as mimics of caddisfly silk. Typically, Poly(SerDPP-co-

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Val) (1.00 g) was dissolved in 50 mL CHCl3: 40% TFA/AcOH (4:1) solution. PtO2 (1.1 eq of

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SerDPP) as a catalyst was added to the polymer solution in a hydrogenation bomb reactor and

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the reaction was carried out in the presence of H2 (60 psi) at room temperature for 2 h. The

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solution was filtered and concentrated under vacuum. The pure polymer was obtained as white

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solid after precipitation in hot water and dried under vacuum.

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5% Poly(pSer-co-Val): 0.9 g, yield = 90%. 1H NMR (500 MHz, DMSO-d6, δ): 0.85 (dd, J =

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6.76 Hz, 13.61 Hz, 194H; CH3), 1.26 (s, 155H, CH2), 1.54 (m, 72H, CH2), 1.98 (td, J = 6.72 Hz,

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13.39 Hz, 35H; CH), 3.87-4.18 (m, 102H, CH2, CH), 4.37 (t, J = 6.48 Hz, 2H; CH2), 6.37 (d, J =

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8.91 Hz, 26H, NH). 13C NMR (500 MHz, DMSO-d6, δ): 19.4 (C4), 25.69 (C4), 28.55 (C4), 30.90

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(C2), 58.15 (C4), 64.53 (C4), 157.97 (NH-C=O), 172.85 (O-C=O). 31P NMR (500 MHz, DMSO-

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d6, 85% H3PO4 external reference, δ): -1.20 ppm. ATR-IR (ν): 1650-1690 cm-1 (s, C=O urea

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stretch), 1735-1750 cm-1 (s, C=O ester stretch), 3300-3500 cm-1 (m, N-H urea stretch), ~1040

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cm-1 (w, P=O stretch), ~710 cm-1 (w, P-O stretch).

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2% Poly(pSer-co-Val): 1 g, yield = 100%. 1H NMR spectra resonances are similar to 5% 13

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Poly(pSer-co-Val) with different integration values.

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spectra corresponds with that of 5% Poly(pSer-co-Val).

C and

31

P NMR spectra and ATR-IR

7


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Surface Energy Measurements. The contact angles of five different liquids were measured to

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calculate the respective surface energy for each of the polymers using the Owens/Wendt method.

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Liquids of known surface tension (water, glycerol, ethylene glycol, propylene glycol, and

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formamide) were used for the contact angle measurements.30 Polymer thin films were spin

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coated on ozone treated silicon wafers using 1% (w/v) solution of the polymers in ethanol at

161

2000 rpm for 1 min. The samples were dried at 70 °C under vacuum overnight. Measurements (n

162

= 3) were performed at room temperature for each sample and the standard deviation of the mean

163

was calculated from independent measurements.

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Lap Shear Adhesion Test On Aluminum Substrate. Lap shear adhesion tests were performed

165

on aluminum substrates according to ASTM D1002 standard. Aluminum adherends 1.6 mm in

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thickness were cut into rectangular substrates 75 mm long x 12.5 mm wide with a 6.5 mm

167

diameter hole drilled 12.5 mm from one end into each adherend. For adhesion tests polymer

168

solution in ethanol (30 µL, 300 mg in 1 mL ethanol) was applied to one end of the adherend.

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Another adherend was placed over it in a lap shear configuration with an overlap area of 1.56

170

cm2. When crosslinkers were utilized, polymer solution (25 µL, 300 mg in 1 mL ethanol) and

171

crosslinker solution (5 µL, 0.3 eq. per pSer group in 1 mL acetone) were mixed and applied to

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one end of the adherend. The adherends were pressed together and allowed to cure for 1 h at

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room temperature and 24 h at 75 °C and 1 h at room temperature before testing. Lap shear

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adhesion was performed using an Instron 5567 instrument with a load cell of 1000 N. The

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adherends were pulled apart at a speed of 1.3 mm/min until failure occurred. The adhesion

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strength (Pa) is obtained by dividing the maximum load at failure (N) by the area of adhesion

177

(m2).

8


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End-To-End Adhesion Test On Bovine Bone Substrate. To study the adhesion characteristics

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of the Poly(pSer-co-Val) we performed end-to-end adhesion experiments on cortical bovine

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bone. Bovine bone samples were obtained from a local grocery store. The bones were cut into

181

rectangular pieces of ~ 2 cm long x 0.6 cm width x 0.4 cm thick on a bone saw. Subsequently,

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the test ends of the bone samples were sanded using a 320 grit sandpaper (3M Pro Grade

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Precision, X-fine) and the bones were kept in PBS solution 1 h prior to adhesive application. The

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polymer solution (30 µL, 300 mg in 1 mL ethanol) was applied on one end of bone and the

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second bone placed end-to-end over it. For crosslinked specimens, polymer solution (25 µL, 300

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mg in 1 mL ethanol) and crosslinker solution (5 µL, 0.3 eq. per pSer group in 1 mL acetone) was

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applied to one bone and the second bone was placed in an end-to-end configuration. The bone

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samples were clipped together and wrapped in PBS soaked gauze for 24 h. The samples were

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then placed in an incubator at 37 °C, 95% humidity and 5% CO2 for 2 h before adhesion tests.

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Adhesion tests were performed on a Texture Analyzer (TA.XT.Plus) equipped with a 5 kg load

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cell. Bone samples were pulled apart at a speed of 1.3 mm/min until failure occurred. The

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adhesion strength (Pa) is obtained by dividing the maximum load at failure (N) by the area of

193

adhesion (m2).

194

In Vitro Cell Viability Study. Cell viability and spreading of MC3T3 cells was studied on the

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phosphate functionalized polymers at day 1 and day 3 respectively after seeding. Polymer films

196

were spun coat from a 3% (w/v) solution in ethanol at 2000 rpm for 1min on a 12 mm glass

197

coverslips. The films were dried overnight at 80 °C under vacuum and carefully transferred to 12

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well plates. Cells were rinsed with PBS prior to detaching with 1mL of 0.05% trypsin/EDTA

199

solution at 37 °C, 95% humidity, 5% CO2 for 5 mins. The trypsin/EDTA solution was

200

deactivated by adding 5 mL of media and cells were collected by centrifugation at 4 °C, 3000 9


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rpm for 1 min. The media/trypsin solution was aspirated without disturbing the cell pellet and

202

cells were resuspended in 5 mL fresh media. The cell density was counted using hemocytometer

203

with trypan blue staining. The cells were seeded at a density of 6000 cells/cm2. The well plates

204

were mildly agitated to ensure even distribution of cells over the samples before incubation.

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Cell viability was assessed by Live/Dead assay (Life Technologies). About 5 µL of Calcein

206

AM (4 mM) and 10 µL of ethidium homodimer were added to 10 mL of DPBS to prepare the

207

staining solution. Media was aspirated from all samples and rinsed with DPBS prior to adding

208

0.5 mL of staining solution to each well. The well plates were covered in aluminum foil and

209

incubated at 37 °C for 10 mins before imaging. Images were taken on an Olympus fluorescence

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microscope equipped with a Hamamatsu orca R2CCD camera, FITC and TRITC filters at 4X

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magnification using CellSENS software. Cells stained green were considered live and the cells

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stained red were considered dead. Cells were counted in ImageJ software using cell counter

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plugin.

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Cell Spreading Assay. For the spreading studies, cells were prefixed in a 1 mL media and 1 mL

215

3.7% paraformaldehyde (PFA) in CS buffer solution for 5 min at 37 °C on a dry block. After

216

aspiration, the cells were fixed in 2 mL 3.7% PFA solution in CS buffer for 5min at 37 °C. The

217

samples were then rinsed 3 times with 2 mL CS buffer followed by addition of 1.5 mL of Triton

218

X-100 in CS buffer (0.5% v/v) in each well to permeabilize the cells for 10 mins at 37 °C. The

219

samples were rinsed 3 times with CS buffer. 2 mL of freshly prepared 0.1 wt% NaBH4 in CS

220

buffer was then added to each well for 10 min and room temperature to quench the aldehyde

221

fluorescence, followed by aspiration and incubation in 5% donkey serum for 20 min at room

222

temperature to block the non-specific binding. After aspiration the samples were incubated in

223

300 µL vinculin primary antibody Mouse in CS buffer (v/v 1:200) at 4 °C overnight. The 10


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samples were then rinsed 3 times with 1% donkey serum and stained with 50 µL rhodamine

225

phalloidin (v/v 1:40) and Alexa Fluor 488 secondary antibody Mouse (v/v 1:200) solution on wax

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paper for 1 h at room temperature in dark. After washing the samples 3 times with CS buffer, the

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nuclei were stained using DAPI in CS buffer (6 µL/10mL) for 20 min at room temperature in

228

dark. The samples were rinsed 3 times with CS buffer to remove excess staining and viewed

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under Olympus fluorescence microscope equipped with a Hamamatsu orca R2CCD camera with

230

FITC, TRITC and DAPI filters at 20X magnification.

231 232

RESULTS AND DISCUSSIONS

233

Synthesis and Characterization of Polymers. Scheme 1 represents the synthesis of

234

Poly(pSer-co-Val). The first step of polymer synthesis involves the solution polymerization of

235

di-p-toluenesulfonic acid salt of bis(L-Valine)-1,8-octanyl diester (1-Val-8) monomer and a

236

diphenyl protected phosphoserine monomer, to produce poly(serine diphenylphosphate-co-

237

valine) (Poly(SerDPP-co-Val)). The second step involves hydrogenolysis deprotection of

238

poly(SerDPP-co-Val) to obtain the phosphate functionalized copolymer, poly(phosphoserine-co-

239

valine) (poly(pSer-co-Val)).

11


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Scheme 1. Synthesis of phosphate functionalized PEU: (a) Copolymerization of di-p-

242

toluenesulfonic acid salt of bis(L-Valine)-1,8-Octanyl-diester (M1) and diphenylphospho serine

243

monomer (M5) in the presence of triphosgene to obtain Poly(SerDPP-co-Val), (b) Deprotection

244

of phenyl protecting groups by hydrogenolysis to obtain Poly(pSer-co-Val).

245 246

In this study, copolymers with 2% and 5% pSer content were synthesized and noted as 2%

247

poly(pSer-co-Val) and 5% poly(pSer-co-Val), respectively.

The actual amount of serine

248

monomer incorporated into the polymer was found to be less than the feed amount as measured

249

by 1H NMR spectroscopy. A plausible justification is the presence of four bulky phenyl groups

250

on the monomer. This tends to lower the reactivity of the serine monomer resulting in low

251

incorporation of serine content. From the 1H NMR spectra of 5% Poly(pSer-co-Val), the phenyl

252

protecting groups show peaks in the aromatic region δ = 7.06-7.34 ppm. The successful

253

deprotection of these phenyl groups after hydrogenolysis was confirmed by the loss of aromatic

254

peaks. The proton environments on the methylene attached directly to the protected phosphate 12


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group have a resonance around δ = 4.0 ppm which shifts slightly downfield to δ = 4.37 ppm

256

after deprotection (Figure 1(a), (b)). The deprotection of diphenyl groups was also confirmed

257

from

258

disappear after deprotection in 5% Poly(pSer-co-Val) (Figure S15 and S16).

259

also verify complete deprotection of the phenyl groups. 5% Poly(SerDPP-co-Val) have a

260

characteristic phosphorus peak at δ = -11.58 ppm which shifts to δ = -1.20 ppm after

261

deprotection (Figure 1(c), (d)).

13

C NMR spectra. The aromatic peaks at δ = ~ 120-130 ppm in 5% Poly(SerDPP-co-Val) 31

P NMR spectra

262

263 264

Figure 1. 1H NMR spectra of (a) 5% Poly(SerDPP-co-Val), (b) 5% Poly(pSer-co-Val); 31P

265

NMR spectra of (c) 5% Poly(SerDPP-co-Val), (d) 5% Poly(pSer-co-Val), referenced to 85%

266

H3PO4 as external standard. Inset shows magnification from δ = 7.1-7.3 ppm of 1H NMR

267

spectra; characteristic peaks of the diphenyl protecting groups disappear after deprotection and δ

268

= 4.0 – 4.5 ppm. A triplet at δ = ~ 4.37 ppm is characteristic of the proton environment on the

269

methylene group attached to the deprotected phosphate group, which is not prominent before

270

deprotection (DMSO-d6, 500 MHz, 30 °C).

13


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The attenuated total internal reflection infrared spectroscopy (ATR-IR), show the presence and

272

conservation of phosphate groups before and after deprotection of the diphenyl groups. The P-O-

273

H bond has a characteristic IR peak in the range of 700–500 cm-1 while P=O bond has a

274

characteristic peak between 1200–1100 cm-1. The P-O characteristic peak for Poly(SerDPP-co-

275

Val) is seen around ~675 cm-1 which shows a slight shift to ~710 cm-1 after deprotection. The

276

P=O bond shows a characteristic peak ~1040 cm-1 before and after deprotection. None of these

277

two characteristic phosphate stretching peaks are prominent in the 1-Val-8 polymer (Poly(1-Val-

278

8)) (Figure S19). The 1H NMR spectra for 2% copolymer resembles that of the 5% copolymer

279

except the integration values are different (Figure S7 and S8). The 13C and 31P NMR spectra for

280

the 2% copolymers also have similar δ (ppm) as that of 5% copolymers (Figure S9 - S12).

281

PEUs were characterized to determine their physical properties like decomposition temperature

282

(Td) by TGA, glass transition temperature (Tg) by DSC, number average molecular weight (Mn),

283

weight average molecular weight (Mw) and polydispersity (ÐM) by SEC. Surface energies of

284

these polymers were determined from contact angles of 5 different liquids of known surface

285

energies to calculate the polar and dispersive components of the polymer surface energy by the

286

following equation29,30:

287

( )

= + .

(1)

288

Where, – Contact angle between polymer and the liquid; – Liquid surface tension; –

289

Dispersive component of liquid surface tension; – Polar component of liquid surface

290

tension; – Polar component of the polymer surface energy; – Dispersive component of

291

polymer surface energy. The total surface energy of the polymer (mJ m-2) is the sum of the

292

polar and dispersive components calculated from Equation 1. Table 1 summarizes the physical 14


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properties of the polymers. The molecular masses of the poly(pSer-co-Val) were not determined

294

to prevent blocking of the SEC columns because of the adhesive nature of the polymer. The

295

hydrogenolysis reaction in the presence of PtO2 catalyst is highly selective and site specific so we

296

did not expect polymer degradation to occur during this reaction.

297 298

Table 1. Physical properties of the poly(ester urea)s

Tg [°C]

a)

Td [°C]

Mn [KDa]

Mw [KDa]

ÐMb)

Surface Energy γP [mJ m-2]c)

Poly(1,8-Val)

19

285

4.3

13.9

3.2

29.0 ± 0.6

2% Poly(SerDPP-co-Val)

8

266

7.7

10.5

1.4

32.4 ± 1.0

5% Poly(SerDPP-co-Val)

5

269

6.5

9.3

1.4

37.0 ± 1.7

2% Poly(pSer-co-Val)

2

175

/

/

/

35.8 ± 0.7

5% Poly(pSer-co-Val)

3

161

/

/

/

33.5 ± 1.5

299 300 301 302

a)Tg determined by DSC; b) ÐM determined from SEC after purification by precipitation in water; c) Surface energy determined by The Owens/Wendt method from contact angle of 5 different liquids on the polymer film.

303

Poly(pSer-co-Val)s show slightly lower glass transition temperatures than poly(SerDPP-co-

304

Val). The presence of the bulky phenyl groups on the protected polymer makes the chains stiff

305

and hinders their movement while after deprotection the chains become more flexible and flow

306

freely showing a slight drop in the respective Tg (Figure S20). Both the protected and

307

deprotected polymers possess decomposition temperatures above 150 °C (Figure S21). The

308

molecular masses of all the polymers are comparable which avoids deviation in adhesion

309

strength due to molecular mass effects. The molecular mass distribution (Dm) is slightly lower 15


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than what is expected of a step growth polymerization reaction for poly(SerDPP-co-Val) because

311

during purification the polymer undergoes fractionation narrowing the Dm. Total surface

312

energies of the polymers along with their polar and dispersive components are tabulated in Table

313

S1. Both the 2% and 5% Poly(pSer-co-Val) have polar and dispersive components for surface

314

energy. The presence of polar component is indicative of surface polar groups that play a

315

significant role in surface adhesion. The surface energies of the 2% and 5% polymers are

316

comparable with each other and are higher than Poly(1-Val-8). Since the percentage of

317

functional groups on the polymers was small we did not see an appreciable change in surface

318

energies between the 2% and 5% functionalization.

319

Lap Shear Adhesion Test on Aluminum Substrate. Lap shear adhesion is a common

320

method used to determine the strength of adhesive materials and their mode of failure. Adhesive

321

strength of the polymer was studied under lap shear configuration at room temperature on

322

aluminum adherends. Lap shear adhesion test was performed on 2% and 5% Poly(pSer-co-Val)

323

with and without crosslinking with Ca2+. Poly(1-Val-8) was used as one of the controls to prove

324

the significance of phosphate groups in adhesion. Commercially available PMMA bone cement

325

was used as another control for comparison with Poly(pSer-co-Val). The results of lap shear

326

adhesion test are summarized in Figure 2(a).

16


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327 328

Figure 2. (a) Lap shear adhesion on aluminum adherends at room temperature. Val – Poly(1-

329

Val-8); 2% and 5% pSer – 2% and 5% Poly(pSer-co-Val) respectively; 2% and 5% Crosslink-

330

2% and 5% Poly(pSer-co-Val) crosslinked with 0.3 eq. of Ca2+ and Bone cement is a

331

commercially available cement (Simplex P). Adhesion strengths were calculated from average of

332

10 replicates (n=10) and reported with standard errors. Aluminum adherends show cohesive

333

failure for all samples, (b) Poly(1-Val-8), (c) 2% Poly(pSer-co-Val), (d) 5% Poly(pSer-co-Val),

334

(e) 2% Poly(pSer-co-Val) crosslinked with 0.3 eq. Ca2+, (f) 5% Poly(pSer-co-Val) crosslinked

335

with 0.3 eq. Ca2+, (g) Poly(methyl methacrylate) bone cement.

336

The adhesion strength of poly(1-Val-8) was 0.04 ± 0.16 MPa and the PMMA bone cement was

337

0.04 ± 0.01 MPa. The low adhesion strength for bone cement is expected because poly(methyl

338

methacrylate) does not have adhesive properties. PMMA bone cements have high modulus and

339

are essentially used as fillers for bone and teeth. Poly(pSer-co-Val) show improved adhesion

340

strength compared to the control samples which could arise from electrostatic or hydrogen

341

bonding interactions between the phosphate groups and the aluminum surface. The adhesion

342

strengths of 2% and 5% poly(pSer-co-Val) are 0.92 ± 0.18 MPa and 0.77 ± 0.09 MPa 17


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343

respectively (Figure 2). Previous studies on caddisfly silk have shown that crosslinking with Ca2+

344

impart strength to the fiber and the crystalline β-sheet structure collapses if the divalent cations

345

are removed suggesting the significance of Ca2+ in the system[22,

346

phosphate groups on the polymer backbone chain will interact with Ca2+ in the crosslinking

347

agent resulting in physical crosslinking which will improve cohesive forces within the polymer

348

bulk further increasing the adhesive strength compared to their uncrosslinked counterparts

349

(Scheme 2). Adhesion strength is the result of combined effects of both adhesive and cohesive

350

forces within a system. To test our hypothesis we chose calcium iodide as the source of Ca2+ for

351

our system.

23]

. Our hypothesis is that

352

Both 2% and 5% Ca2+ crosslinked poly(pSer-co-Val) showed an increase in the adhesion

353

strengths compared to their uncrosslinked counterparts. The adhesion strengths of crosslinked

354

2% poly(pSer-co-Val) and crosslinked 5% poly(pSer-co-Val) are 1.17 ± 0.19 MPa and 1.14 ±

355

0.02 MPa respectively (Figure 5). Our results demonstrate the strong affinity of the phosphate

356

functionalized polymers on the aluminum substrates. Cohesive failure is when the adhesive is

357

stuck on both the adherends after failure. If the adhesive cleanly detaches from one adherend and

358

sticks to the other after failure, it is called adhesive failure. The mode of failure is important in

359

determining the commercial potential of an adhesive. For most adhesives, cohesive failure is

360

desirable. Interestingly, all the samples exhibit cohesive failure except the bone cement which

361

failed adhesively (Figure 2(b) to (g). This result is not surprising considering the fact that bone

362

cements are not adhesive in nature but only serve as a support medium for the bone. The results

363

of the adhesion studies on metal suggests that incorporation of even small percentage of

364

phosphate groups on the polymer backbone show significant improvement in the adhesion

18


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strength on metal substrate. Also, crosslinking of the phosphate groups with divalent cations

366

further enhances the adhesion strength via electrostatic interaction.

367 368

Scheme 2. Phosphate groups ( ) on Poly(pSer-co-Val) interact with positive charges on the

369

bone surface promoting adhesion to the bone. Ca2+ from calcium iodide (cross-linking agent)

370

interact with phosphate groups in the bulk of the polymer giving rise to cohesive forces.

371

End-to-end Adhesion Test on Bovine Bone. We also tested the adhesion strengths of

372

poly(pSer-co-Val) on bovine bone samples to demonstrate its potential application as a bone

373

adhesive. Bones have an array of positive and negative charges on the surface. The basic

374

building block of bone is hydroxyapatite with the chemical structure of Ca5(PO4)3(OH). Figure

375

3(a) summarizes the adhesion strengths of the polymers on bovine bone. The controls for this

376

study were the same as that for the metal substrate. Poly(1-Val-8) had absolutely no adhesion to

377

the bone surface. All the samples failed before performing the tests. Poly(pSer-co-Val) show

378

increased adhesion strengths compared to Poly(1-Val-8) control. 2% Poly(pSer-co-Val) showed

379

adhesion strength of 190 ± 70 KPa and 5% Poly(pSer-co-Val) showed adhesion strength of 399

380

± 101 KPa. 19


Biomacromolecules

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381 382

Figure 3. (a) End-to-end adhesion on bovine bone at room temperature. Val – Poly(1-Val-8); 2%

383

and 5% pSer – 2% and 5% Poly(pSer-co-Val) respectively; 2% and 5% Crosslink- 2% and 5%

384

Poly(pSer-co-Val) crosslinked with 0.3 eq. of Ca2+, Bone cement – commercially available

385

PMMA cement (Simplex P). Adhesion strengths were calculated from average of 3 replicates

386

(n=3) and reported with standard errors, (b) Schematic of end-to-end adhesion on bovine bone

387

sample (Left) and end-to-end adhesion test on texture analyzer (Right), (c) Image showing

388

cohesive failure of 5% Poly(pSer-co-Val) crosslinked with 0.3 eq. of Ca2+.

389

An increase in adhesion strength was observed after adding 0.3 eq. of Ca2+ as crosslinking

390

agent. The adhesion strength of crosslinked 2% Poly(pSer-co-Val) increased to 211 ± 77 KPa

391

while that of crosslinked 5% Poly(pSer-co-Val) increased to 439 ± 203 KPa. The increase in

392

adhesion strength of the phosphate functionalized polymer compared to that of the control

393

polymer proves the significance of the presence of phosphate groups. It is also notable that

394

incorporation of a small percentage of phosphate functionality brings an appreciable change in 20


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395

adhesion. The adhesion strength of 5% poly(pSer-co-Val) is comparable to commercially

396

available bone cement (530 ± 133 KPa) which suggests that poly(pSer-co-Val) has strong

397

potential for further development into bone adhesives. All the samples showed cohesive failure

398

except for the bone cement which showed adhesive failure.

399

In Vitro Cell Viability and Spreading Assay. The cell viability of MC3T3 cells was

400

calculated using atleast 40 images for each sample and normalized to 2% Poly(pSer-co-Val)

401

(Figure 4(a)-(f)). The viability of cells on glass, poly(1-Val-8), 2% and 5% poly(pSer-co-Val),

402

2% and 5% crosslinked poly(pSer-co-Val) was (84 ± 9)%, (94 ± 8)%, (100 ± 3)%, (97 ± 5)%,

403

(97 ± 7)% and (98 ± 4)% respectively (Figure S22). The low cell viability on glass could be

404

attributed to handling and seeding errors. High cell viabilities (>95%) on the functionalized

405

polymers prove that the phosphate functionality is non-toxic to the cells. The cytoskeletal

406

structure or spreading behavior of MC3T3 cells on all samples was similar (Figure 4(g)-(l)). The

407

cells were stained blue (DAPI) for nuclei, green (Alexa Fluor 488) for focal adhesion points and

408

rhodamine phalloidin (red) for actin filaments. The aspect ratio of the cells on all samples were

409

fairly close (~ 2-3), which proves that the functionalized polymers behaved similar to the

410

controls (Figure S23).

411 412

Figure 4. Cell Viability ((a)-(f)) and Spreading analysis of MC3T3 ((g)-(l)) cells on day 1 and

413

day 3 respectively. (a) and (g) Glass substrate, (b) and (h) Poly(1-Val-8), (c) and (i) 2% 21


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414

Poly(pSer-co-Val), (d) and (j) 5% Poly(pSer-co-Val), (e) and (k) 2% Poly(pSer-co-Val)

415

crosslinked with 0.3 eq. Ca2+, (f) and (l) 5% Poly(pSer-co-Val) crosslinked with 0.3 eq. Ca2+.

416

Cell viability on different samples was studied by live-dead assay from atleast 40 images per

417

sample (4x magnification, scale bar ~ 200 µm). Live cells were stained green by calcein AM and

418

dead cells were stained red by ethidium homodimer. For spreading studies cells were stained

419

with rhodamine phalloidin – actin filaments (red), alexa fluor 488 secondary antibody – focal

420

adhesion points (green) and dapi – nuclei (blue). Images were taken at 20x magnification (Scale

421

bar ~ 50 µm) and aspect ratios were calculated from 30 cells per sample.

422

CONCLUSION

423

Phosphate functionalized PEU copolymers were designed and created to mimic the properties

424

of caddisfly adhesive silk. These copolymers are ethanol soluble which provides a suitable

425

delivery mechanism and makes them suitable for medical applications. The copolymers showed

426

maximum adhesion strength of 1.17 ± 0.19 MPa on metal substrates after crosslinking with Ca2+.

427

The adhesive strengths of copolymers on bone samples were significant (439 ± 203 KPa) and

428

comparable to commercially available poly(methyl methacrylate) bone cement (530 ± 133 KPa).

429

The phosphate functionalized copolymers demonstrated improved and significant adhesion

430

strength compared to the valine polymer analogs demonstrating that the phosphate groups play a

431

key role in promoting adhesion. In all cases the copolymers showed cohesive failure. The

432

phosphate functionalized copolymers have significant potential as orthopaedic adhesives,

433

scaffold materials for spinal cord injury and orthopaedic repairs in the presence of growth

434

peptides like OGP or BMP-2. PEUs are degradable in vitro and in vivo. Our future efforts will

435

focus on improving the pSer content in the copolymers, studying the effect of varying the

436

pSer:Ca2+ ratio, curing kinetics and adhesive properties on translationally relevant substrates. 22


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437

ASSOCIATED CONTENT

438

Supporting Information.

439

Detailed synthesis of monomers. 1H,

13

C and

31

P NMR spectra of all the monomers and

440

polymers, DSC and TGA curves of polymers, ATR-IR spectra of polymers, Graphical

441

representation of cell viability and cell spreading on different polymers, Polar and dispersive

442

surface energies of all polymers. This material is available free of charge via the Internet at

443

http://pubs.acs.org.

444

AUTHOR INFORMATION

445

Corresponding Author

446

*Email: [email protected]. Phone:

447

Present Addresses

448

† State Key Laboratory of High Performance Civil Engineering Materials, Nanjing, Jiangsu,

449

China, 211103

450

Author Contributions

451

The manuscript was written through contributions of all authors. All authors have given approval

452

to the final version of the manuscript.

Fax:

453 454

ACKNOWLEDGMENT

455

Funding from the Biomaterials Division of the National Science Foundation (DMR-1507420)

456

and the Ohio Department of Development is gratefully acknowledged. The authors would like to 23


Biomacromolecules

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457

thank Dr. Rajarshi Sarkar and Dr. Charles Moorefield for their assistance in hydrogen reduction

458

reactions. E.O. was funded by a summer NSF REU award (DMR-1359321).

459

REFERENCES

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16. Lipik, V.; Zhang, L. H.; Miserez, A., Synthesis of biomimetic co-polypeptides with tunable degrees of phosphorylation. Polym Chem-Uk 2014, 5, (4), 1351-1361. 17. Shao, H.; Stewart, R. J., Biomimetic underwater adhesives with environmentally triggered setting mechanisms. Adv Mater 2010, 22, (6), 729-33. 18. Breucker, L.; Landfester, K.; Taden, A., Phosphonic Acid-Functionalized Polyurethane Dispersions with Improved Adhesion Properties. ACS Appl Mater Interfaces 2015, 7, (44), 24641-8. 19. Stewart, R. J., Protein-based underwater adhesives and the prospects for their biotechnological production. Appl Microbiol Biot 2011, 89, (1), 27-33. 20. Yonemura, N.; Mita, K.; Tamura, T.; Sehnal, F., Conservation of silk genes in Trichoptera and Lepidoptera. J Mol Evol 2009, 68, (6), 641-53. 21. Wang, Y.; Sanai, K.; Wen, H.; Zhao, T.; Nakagaki, M., Characterization of unique heavy chain fibroin filaments spun underwater by the caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae). Mol Biol Rep 2010, 37, (6), 2885-92. 22. Addison, J. B.; Ashton, N. N.; Weber, W. S.; Stewart, R. J.; Holland, G. P.; Yarger, J. L., beta-Sheet nanocrystalline domains formed from phosphorylated serine-rich motifs in caddisfly larval silk: a solid state NMR and XRD study. Biomacromolecules 2013, 14, (4), 1140-8. 23. Addison, J. B.; Weber, W. S.; Mou, Q.; Ashton, N. N.; Stewart, R. J.; Holland, G. P.; Yarger, J. L., Reversible assembly of beta-sheet nanocrystals within caddisfly silk. Biomacromolecules 2014, 15, (4), 1269-75. 24. Stewart, R. J.; Wang, C. S., Adaptation of caddisfly larval silks to aquatic habitats by phosphorylation of h-fibroin serines. Biomacromolecules 2010, 11, (4), 969-74. 25. Wang, C. S.; Ashton, N. N.; Weiss, R. B.; Stewart, R. J., Peroxinectin catalyzed dityrosine crosslinking in the adhesive underwater silk of a casemaker caddisfly larvae, Hysperophylax occidentalis. Insect Biochem Mol Biol 2014, 54, 69-79. 26. Wang, C.-S.; Pan, H.; Weerasekare, G. M.; Stewart, R. J., Peroxidase-catalysed interfacial adhesion of aquatic caddisworm silk. Journal of The Royal Society Interface 2015, 12, (112). 27. Kartvelishvili, T.; Tsitlanadze, G.; Edilashvili, L.; Japaridze, N.; Katsarava, R., Amino acid based bioanalogous polymers. Novel regular poly(ester urethane)s and poly(ester urea)s based on bis(L-phenylalanine) α,ω-alkylene diesters. Macromol Chem Phys 1997, 198, (6), 1921-1932. 28. Zimmermann, J.; Loontjens, T.; Scholtens, B. J.; Mulhaupt, R. R., The formation of poly(ester-urea) networks in the absence of isocyanate monomers. Biomaterials 2004, 25, (14), 2713-9. 29. Stakleff, K. S.; Lin, F.; Smith Callahan, L. A.; Wade, M. B.; Esterle, A.; Miller, J.; Graham, M.; Becker, M. L., Resorbable, amino acid-based poly(ester urea)s crosslinked with osteogenic growth peptide with enhanced mechanical properties and bioactivity. Acta Biomater 2013, 9, (2), 5132-42. 30. Yu, J. Y.; Lin, F.; Lin, P. P.; Gao, Y. H.; Becker, M. L., Phenylalanine-Based Poly(ester urea): Synthesis, Characterization, and in vitro Degradation. Macromolecules 2014, 47, (1), 121129. 31. Zhou, J.; Defante, A. P.; Lin, F.; Xu, Y.; Yu, J.; Gao, Y.; Childers, E.; Dhinojwala, A.; Becker, M. L., Adhesion properties of catechol-based biodegradable amino acid-based poly(ester urea) copolymers inspired from mussel proteins. Biomacromolecules 2015, 16, (1), 266-74. 25


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Caddisfly Inspired Phosphorylated Poly(ester urea)-Based

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