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Article
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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Biomacromolecules
1
Caddisfly Inspired Phosphorylated Poly(ester-urea)
2
based Degradable Bone Adhesives
3 4 5
Vrushali Bhagat, Emily O’Brien, Jinjun Zhou† and Matthew L. Becker* Department of Polymer Science, The University of Akron, Akron, OH-44325, USA
6
ABSTRACT
7
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
9
adhesion strengths that degrade into bioresorbable byproducts. Caddisfly adhesive silk is
10
interesting due to the presence of phosphoserines which are thought to afford adhesive
11
properties. In this work phosphoserine-valine poly(ester urea) copolymers with 2% and 5%
12
phosphoserine content were synthesized to mimic caddisfly adhesive silk. Significantly the
13
materials are ethanol soluble and water insoluble making them clinically relevant. Their physical
14
properties were quantified and the adhesion properties were studied on aluminum and bovine
15
bone substrates before and after crosslinking with Ca2+ ions. The adhesive strength of the
16
phosphorylated copolymer on a bone substrate after crosslinking with Ca2+ was 439 ± 203 KPa,
17
comparable to commercially available PMMA bone cement (530 ± 133 KPa).
18
KEYWORDS: Caddisfly, poly(ester-urea), phosphoserine, tissue adhesives, adhesion strength
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INTRODUCTION
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Bone and tissue adhesives are essential in surgeries for their assistance in wound healing,
22
hemostasis, tissue reconstruction and drug delivery.1 Classical structural alternatives in bone
23
surgeries include use of metallic plates, pins and screws as support medium. These options while
24
safe and serviceable suffer from aseptic loosening, poor anchorage to the bone, cause irritation to
25
the neighboring soft tissue, cause discomfort to the patient and need to be replaced or removed
26
after the bone regeneration. Poly(methyl methacrylate) (PMMA) bone cement is also commonly
27
used in bone surgeries however, it is not adhesive in nature, lack chemical interaction, cause
28
significant heat generation and shrinkage. Attempts to use synthetic glues like cyanoacrylate,
29
polyurethane, epoxy resin and calcium or magnesium phosphate ceramic bone cement as bone
30
adhesives have failed either due to lack of interaction with the bone surface or poor strength.2
31
Alternative, bone adhesives with degradable properties and high adhesion strengths are currently
32
an attractive clinical target.
33
Phosphate based compounds have been used as adhesion promoters for decades in underwater
34
coatings, dental applications, bone implants, fillers and metal substrates.3-8 Several studies have
35
demonstrated adhesion or bonding strength and osteoconductive potential of phosphate
36
functionalized polymeric bone grafts showing significant improvement in bone bonding.5,
37
Recently Breucker et.al. developed a facile route to phosphorus functionalized polyurethane
38
aqueous dispersions. Quartz crystal microbalance (QCM) studies showed enhanced affinity of
39
the dispersions towards hydroxyapatite and stainless steel surfaces suggesting their application as
40
potential bone adhesives.18 Clearly, phosphate containing polypeptides or polymers demonstrate
41
improved adhesion behavior on hydroxyapatite or bone surface and show promise for next
42
generation bone adhesives.
9-17
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Marine organisms like mussels, barnacles, starfish, sanscastle worms and caddisflies have
44
evolved to synthesize their own underwater adhesive with strong bonding characteristics.19
45
Studies on caddisfly adhesive silk have confirmed the presence of phosphoserine (pSer) residues
46
in the form of (SX)4 motifs where S is serine and X is usually valine or isoleucine. Elemental
47
analysis also showed the presence of divalent cations like Ca2+ and Mg2+ which undergo strong
48
electrostatic interaction with the phosphate groups in pSer to impart strength to the fibers. The
49
adhesive nature of caddisflies is attributed to the heavily phosphorylated regions in the adhesive
50
filament.20-24 Recent studies by Wang et.al. describe an alternative model of caddisfly silk
51
adhesion consisting of a crosslinked dityrosine peripheral layer and an evenly distributed PEVK
52
like protein throughout the fiber core. The dityrosines crosslinked in the presence of reactive
53
oxygen species were thought to impart strength and adhesion to the fibers.25, 26 However, the role
54
of phosphate groups as adhesion promoters in caddisfly silk was not completely ruled out. In this
55
work, inspired from the caddisfly adhesive silk, a phosphate functionalized poly(ester urea) was
56
created as a bone adhesive.
57
Poly(ester urea)s (PEU)s are a class of polymers well suited for biomaterial applications
58
because of their attractive properties including degradation into metabolic components, tunable
59
mechanical properties, wide range of functionality and nontoxicity in vitro and in vivo.27-31 PEU
60
copolymers based on pSer and valine amino acid, as found in caddisfly silk were synthesized and
61
characterized foe their physical properties. The pSer content incorporated in the copolymer was
62
2% and 5%. The solubility of these polymers in ethanol makes them more clinically relevant and
63
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
65
substrates and end-to-end adhesion on bovine bone substrates. 3
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EXPERIMENTAL SECTION
68
Materials and Methods. L-valine, p-toluene sulfonic acid monohydrate, toluene, 1,8-octanediol,
69
anhydrous dichloromethane (DCM), Pd/C, PtO2, ethanol, diphenylphosphoryl chloride (99%),
70
anhydrous pyridine, 4M HCl/Dioxane solution, chloroform, trimethylamine and acetic acid were
71
purchased from Sigma Aldrich. N-Boc-O-Bzl-L-Serine was purchased from Ark Pharm, Inc.
72
N,N-Diisopropyl carbodiimide (DIC) was purchased from Oakwood Chemicals. Triphosgene
73
was purchased from TCI America. 4-(dimethyl amino) pyridinium 4-toluenesulfonate (DPTS)
74
was synthesized according to literature procedure. Chloroform was dried over CaH2 overnight,
75
distilled and stored in dark before use. All chemicals were used as received unless noted
76
otherwise.
77
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
79
ppm and referenced to residual solvent resonances (1H NMR, DMSO-d6: δ = 2.50 ppm and 13C
80
NMR, DMSO-d6: δ = 39.50 ppm). Abbreviations of multiplicities are denoted as s-singlet, d-
81
doublet, m-multiplet, q-quartet, dd-double doublet, td-triple doublet. Attenuated Total Internal
82
Reflection Infrared (ATR-IR) Spectra of polymers were recorded on Shimadzu Miracle 10 ATR-
83
FTIR equipped with a quartz crystal window. A small piece of polymer, enough to cover the
84
crystal window was used for the measurements. Electrospray Ionization Mass spectrum (ESI-
85
MS) of monomer was recorded on Bruker HCTultra II quadrupole ion trap (QIT) mass
86
spectrometer (Billerica, MA) equipped with an ESI source. Molecular weights and PDI (ÐM) of
87
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
89
equipped with a refractive index detector. The glass transition temperature (Tg) of polymers was
90
determined by Differential Scanning Calorimetry (DSC, TA Q200) at a scanning rate of 10
91
°
92
determined using Thermogravimetric Analysis (TGA, TA Q500) at a heating rate of 10 °C/min
93
from 25 °C to 600 °C.
94
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.
96
Synthesis
of
1,8-octanediol-L-valine
poly(ester
urea)
(Poly(1-Val-8)):
A
solution
97
polymerization was carried out by dissolving the di-p-toluenesulfonic acid salt of bis(L-Valine)-
98
1,8-Octanyl-diester (M1) (13.74 g, 20 mmol, 1 eq.) and triethylamine (12.55 ml, 90 mmol, 4.5
99
eq.) in 40 mL of chloroform in a 500 mL three neck round bottom flask equipped with a
100
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
102
nitrogen at room temperature. The reaction continued for 12 h after which an additional aliquot
103
of triphosgene (0.45 g, 1.5 mmol, 0.08 eq.) dissolved in 15 mL of chloroform was added to the
104
flask dropwise. The reaction was stirred for an additional 8 h. The polymer was purified by
105
precipitation in hot water and dried under vacuum. 1H NMR (500 MHz, DMSO-d6, δ): 0.86 (m,
106
12H, CH3), 1.26 (s, 8H, CH2), 1.54 (s, 4H, CH2), 1.98 (dd, J = 6.69 Hz, 11.73 Hz, 2H; CH),
107
3.78-4.21 (m, 6H, CH2, CH), 6.37 (d, J = 8.89 Hz, 2H; NH). 13C NMR (500 MHz, DMSO-d6, δ):
108
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,
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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
111
KDa.
112
Synthesis
of
Poly(serine
diphenylphosphate-co-valine)
(Poly(SerDPP-co-Val):
The
113
copolymer was synthesized via a solution polymerization similar to P(1-Val-8). Typically, both
114
the monomers (20 mmol, 1 eq.) and triethylamine (90 mmol, 4.5 eq.) were dissolved in 40 mL of
115
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
118
12 h and then additional aliquot of triphosgene (1.5 mmol, 0.075 eq in 10 mL of chloroform) was
119
added dropwise to the flask. The reaction continued for another 8 h after which the reaction
120
solution was transferred to a separatory funnel and precipitated dropwise in hot water. After the
121
water cools to ambient temperature, the polymer was washed in water at room temperature
122
overnight and finally dried under vacuum to obtain white polymer as product.
123
5% Poly(SerDPP-co-Val): Theoretical monomer feed ratio- pSer/Val (M5/M1) = 15/85: 1H
124
NMR (500 MHz, DMSO-d6, δ): Actual Monomer ratio = 5/95, δ = 0.85 (dd, J = 6.76 Hz, 13.63
125
Hz, 195H; CH3), 1.26 (d, J = 11.12 Hz, 154H; CH2), 1.54 (m, 72H, CH2), 1.98 (dd, J = 6.56 Hz,
126
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
127
(m, 26H, Ar H), 7.19-7.34 (m, 12H, Ar H). 13C NMR (500 MHz, DMSO-d6, δ): 19.4 (C4), 25.69
128
(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).
130
reference, δ): 11.58 ppm. ATR-IR (ν): 1650-1690 cm-1 (s, C=O urea stretch), 1735-1750 cm-1 (s,
131
C=O ester stretch), 3300-3500 cm-1 (m, N-H urea stretch), ~1040 cm-1 (w, P=O stretch), ~675
132
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
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2% Poly(SerDPP-co-Val): Theoretical monomer feed ratio- pSer/Val (M5/M1) = 10/90: 1H
134
NMR spectra resonances are similar to 5% P(SerDPP-co-Val) with different integration values.
135
Actual Monomer ratio = 2/98. 13C and 31P NMR spectra and ATR-IR spectra corresponds with
136
that of 5% Poly(SerDPP-co-Val). ÐM = 1.4, Mn = 6.5 KDa, Mw = 9.3 KDa.
137
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
139
phosphate functionalized copolymers as mimics of caddisfly silk. Typically, Poly(SerDPP-co-
140
Val) (1.00 g) was dissolved in 50 mL CHCl3: 40% TFA/AcOH (4:1) solution. PtO2 (1.1 eq of
141
SerDPP) as a catalyst was added to the polymer solution in a hydrogenation bomb reactor and
142
the reaction was carried out in the presence of H2 (60 psi) at room temperature for 2 h. The
143
solution was filtered and concentrated under vacuum. The pure polymer was obtained as white
144
solid after precipitation in hot water and dried under vacuum.
145
5% Poly(pSer-co-Val): 0.9 g, yield = 90%. 1H NMR (500 MHz, DMSO-d6, δ): 0.85 (dd, J =
146
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,
147
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 =
148
8.91 Hz, 26H, NH). 13C NMR (500 MHz, DMSO-d6, δ): 19.4 (C4), 25.69 (C4), 28.55 (C4), 30.90
149
(C2), 58.15 (C4), 64.53 (C4), 157.97 (NH-C=O), 172.85 (O-C=O). 31P NMR (500 MHz, DMSO-
150
d6, 85% H3PO4 external reference, δ): -1.20 ppm. ATR-IR (ν): 1650-1690 cm-1 (s, C=O urea
151
stretch), 1735-1750 cm-1 (s, C=O ester stretch), 3300-3500 cm-1 (m, N-H urea stretch), ~1040
152
cm-1 (w, P=O stretch), ~710 cm-1 (w, P-O stretch).
153
2% Poly(pSer-co-Val): 1 g, yield = 100%. 1H NMR spectra resonances are similar to 5% 13
154
Poly(pSer-co-Val) with different integration values.
155
spectra corresponds with that of 5% Poly(pSer-co-Val).
C and
31
P NMR spectra and ATR-IR
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Surface Energy Measurements. The contact angles of five different liquids were measured to
157
calculate the respective surface energy for each of the polymers using the Owens/Wendt method.
158
Liquids of known surface tension (water, glycerol, ethylene glycol, propylene glycol, and
159
formamide) were used for the contact angle measurements.30 Polymer thin films were spin
160
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.
164
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
166
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.
169
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
172
one end of the adherend. The adherends were pressed together and allowed to cure for 1 h at
173
room temperature and 24 h at 75 °C and 1 h at room temperature before testing. Lap shear
174
adhesion was performed using an Instron 5567 instrument with a load cell of 1000 N. The
175
adherends were pulled apart at a speed of 1.3 mm/min until failure occurred. The adhesion
176
strength (Pa) is obtained by dividing the maximum load at failure (N) by the area of adhesion
177
(m2).
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End-To-End Adhesion Test On Bovine Bone Substrate. To study the adhesion characteristics
179
of the Poly(pSer-co-Val) we performed end-to-end adhesion experiments on cortical bovine
180
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,
182
the test ends of the bone samples were sanded using a 320 grit sandpaper (3M Pro Grade
183
Precision, X-fine) and the bones were kept in PBS solution 1 h prior to adhesive application. The
184
polymer solution (30 µL, 300 mg in 1 mL ethanol) was applied on one end of bone and the
185
second bone placed end-to-end over it. For crosslinked specimens, polymer solution (25 µL, 300
186
mg in 1 mL ethanol) and crosslinker solution (5 µL, 0.3 eq. per pSer group in 1 mL acetone) was
187
applied to one bone and the second bone was placed in an end-to-end configuration. The bone
188
samples were clipped together and wrapped in PBS soaked gauze for 24 h. The samples were
189
then placed in an incubator at 37 °C, 95% humidity and 5% CO2 for 2 h before adhesion tests.
190
Adhesion tests were performed on a Texture Analyzer (TA.XT.Plus) equipped with a 5 kg load
191
cell. Bone samples were pulled apart at a speed of 1.3 mm/min until failure occurred. The
192
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
195
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
198
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.
205
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
210
microscope equipped with a Hamamatsu orca R2CCD camera, FITC and TRITC filters at 4X
211
magnification using CellSENS software. Cells stained green were considered live and the cells
212
stained red were considered dead. Cells were counted in ImageJ software using cell counter
213
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
227
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
229
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)).
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240 241
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).
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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).
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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
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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
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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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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
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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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