Polyurethane (PU)-derived photoactive and copper-free clickable surface based on perfluorophenyl azide (PFPA) chemistry

Article abstract of DOI:10.1039/c2tb00248e

The anchoring and capturing roles of perfluorophenyl azide (PFPA) were combined to produce a universal polyurethane (PU)-derived photoactive surface platform (PU-1-PFPA). The resultant platform was confirmed by contact angle, attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) analyses. Upon UV light activation, native heparin was coupled directly onto PU-1-PFPA to yield a substrate with antithrombogenic properties. The same level of antithrombogenic activity was achieved when the recovered heparin was photo-coupled onto PU-1-PFPA. In addition, at room temperature and in the absence of copper catalysts, PU-1-PFPA achieved oriented immobilization of functional moieties bearing an alkynyl functional group.

Full text of DOI:10.1039/c2tb00248e

Journal of
Materials Chemistry B
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PAPER
Polyurethane (PU)-derived photoactive and copper-free
clickable surface based on perfluorophenyl azide (PFPA)
chemistry†
Cite this: J. Mater. Chem. B, 2013, 1,
71
5
Lingdong Li, Jiang Li, Abhilash Kulkarni and Song Liu*
The anchoring and capturing roles of perfluorophenyl azide (PFPA) were combined to produce a universal
polyurethane (PU)-derived photoactive surface platform (PU-1-PFPA). The resultant platform was
confirmed by contact angle, attenuated total reflectance Fourier transform infrared (ATR FT-IR)
spectroscopy, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) analyses.
Upon UV light activation, native heparin was coupled directly onto PU-1-PFPA to yield a substrate with
antithrombogenic properties. The same level of antithrombogenic activity was achieved when the
recovered heparin was photo-coupled onto PU-1-PFPA. In addition, at room temperature and in the
absence of copper catalysts, PU-1-PFPA achieved oriented immobilization of functional moieties bearing
an alkynyl functional group.
Received 12th October 2012
Accepted 14th November 2012
DOI: 10.1039/c2tb00248e
www.rsc.org/MaterialsB
radical and the primary radical generated from the initiator
produces a covalent attachment of the initiator molecule.
Introduction
Synthetic polymeric materials such as poly(ethylene tere- Benzophenone, a typical Norrish II initiator, has been success-
8
9
phthalate) (PET), polyurethane (PU), and poly(lactic acid) (PLA) fully graed onto poly(dimethylsiloxane), polyethylene, poly-
10
11
have been widely used for medical applications including propylene, and PET. If benzophenone is derivatized with
11
vascular gras, ligament and tendon prostheses, and tissue functional groups, direct functionalization of the substrate
1
,2
engineering scaffolds. However, oen these materials do not
polymer could be achieved. Upon exposure to UV irradiation
365 nm), the benzophenone moiety anchors to the surface of
bulk polymers so as to introduce new functional groups. Photo-
have the appropriate surface properties essential for inciting in
situ tissue regeneration or for avoiding adverse biological
responses such as thrombosis and inammatory response.
(
3
4
generated carbenes and nitrenes preferably insert into C–H, N–H
Therefore, bioactive agents are usually required to be immobi- and C]C to form covalent bonds. Two typical corresponding
12
lized on material surfaces to help meet the biological challenges photoreagents are triuoromethyl-aryldiazirine
at the tissue–biomaterial interface.
and per-
uorophenyl azide (PFPA) and these two photoreagents can also
On surfaces with functional groups such as hydroxyl and be derivatized with to-be-immobilized target functional groups.
13
amine groups, many coupling agents can be easily introduced to
Aer the introduction of functional groups onto inert poly-
meric substrates using photoimmobilization strategies,
secondary chemistry is needed for the covalent bonding of
serve as linker molecules for the eventual immobilization of the
5–7
bioactive compounds. However, the choice of a coupling agent
universally applicable to a wide range of polymeric substrates is biomolecules. For example, aldehyde groups were introduced
relatively limited. The universal chemistry for covalent surface onto the surface of nylon membranes by photoactivating
modication of polymeric substrates is generally based on the p-azidobenzaldehyde coated on the membrane, and subse-
generation of reactive radicals (from Norrish II initiators), car- quently various antibodies were covalently bound through a
14
benes and nitrenes. Radicals generated from Norrish type II reaction with the surface aldehyde groups. The photosensitive
initiators have a high tendency to abstract hydrogen from
substrate polymers. The subsequent coupling of the substrate
heads of the three photoreagents, i.e. benzophenone, tri-
uoromethyl aryldiazirine and peruorophenyl azide (PFPA),

can also covalently capture biomolecules onto the surfaces if
these photoreagents are rst immobilized via thermochemical
Department of Textile Sciences, Faculty of Human Ecology, Department of Chemistry, reactions. For instance, PFPA has been immobilized on silicon
Faculty of Science, University of Manitoba, Winnipeg, Canada R3T 2N2. E-mail: Song.
Liu@ad.umanitoba.ca; Fax: +1 204-474-7593; Tel: +1 204-474-9616
15
16,17
oxide and metal nanoparticles
using silane chemistry and
transition metal sulfur chemistry respectively, resulting in
photoactive surfaces for secondary functionalization.
The combination of photo-anchoring and photo-capturing
has attracted interest from investigators for some time. Sigrist
†
Electronic supplementary information (ESI) available: Synthesis attempt on
compound 4 and characterization of PU-3 including ATR, AFM and calculation
of surface functionalization level, and the self-click phenomenon on a TLC
plate between PFPA and alkynes. See DOI: 10.1039/c2tb00248e
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18
and coworkers reported a one-step photoimmobilization of of integrating surface functionalities. Yan and coworkers have
biomolecules (streptavidin, inulin, and encephalin) onto poly- successfully photoanchored 2,5-dioxopyrrolidin-1-yl-4-azido-
29
styrene in the presence of triuoromethyl aryldiazirine deriv- 2,3,5,6-tetrauorobenzoate (PFPA-NHS (1)) on polystyrene (PS)
atized bovine serum albumin (BSA) whose degree of to give the surface an NHS-platform, onto which amine con-
photolabeling was 8–10 mol triuoromethyl aryldiazirine per taining biomolecules can be coupled. Proteins and oligonucle-
mol BSA. Upon light activation, some of the generated carbenes otides have been covalently attached onto the NHS-platform for
30,31
along the backbone of BSA insert into the polystyrene substrate biological sensing and imaging applications,
but this
while the others insert into the biomolecules, producing a methodology does not work in the case of amine-free target
covalent graing of the biomolecules onto the chemically inert bioactive reagents such as heparin. We proposed to confer
19
polystyrene. Pei et al. realized a carbohydrate microarray PFPA-amine (2) onto the NHS-platform to afford a surface
through multi-steps of photo-capturing and photo-anchoring: photoactive platform (SPP) onto which underivatized functional
following the photo-capturing of poly(ethylene oxide) onto PFPA cargoes of interest can be photo-coupled.
functionalized glass slides, PFPA derivatized carbohydrates
In this work, we photo-coupled 1 onto a PU lm (PU-1) and
were photo-anchored onto the surface in an arrayed manner. subsequently attached 2 onto PU-1 to fabricate the general SPP
Although there is a trend to incorporate both photo-anchoring as PU-1-PFPA (Scheme 1). The modied surface was character-
18,19
and photo-capturing processes in surface ligation,
the ized using contact angle measurements, attenuated total
combination of PFPA photo-anchoring/capturing strategy to reectance Fourier transform infrared (ATR FT-IR) spectroscopy,
covalently immobilize functional moieties onto thermoplastic atomic force microscopy (AFM) and X-ray photoelectron spec-
polymers lacking reactive groups has not been explored.
troscopy (XPS). Without chemical derivatization, heparin was
Photoimmobilization strategies are extremely useful to successfully photo-immobilized onto PU-1-PFPA using UV irra-
introduce reactive functions on chemically inert surfaces and to diation. Platelet adhesion to the resultant PU lm was proven to
capture biomolecules lacking reactive functional groups. be signicantly less than that to the untreated PU lm. The
However, the abovementioned photoimmobilization chemistry constructed platform (PU-1-PFPA) can not only capture heparin
suffers from one problem: it lacks functional group specicity. photochemically, but can also conjugate with alkynyl functional
Nevertheless, the photoreagent PFPA carries an azide functional moieties in the absence of toxic copper catalysts.
group which has been reported to react orthogonally and
quantitatively with an alkyl group under the catalysis of cop-
Experimental section
Materials and agents
20
per(I). So, we hypothesize that we can build a versatile and
exible surface platform for both the immobilization of bioac-

tive agents lacking reaction groups and the oriented immobi- The solvents and chemicals such as methyl penta-
lization of alkynyl functional moieties by combining the uorobenzoate, 2-propynylamine, 2,2-methylbut-3-yn-1-amine,
photoreactivity and click potential of PFPA.
4-chlorobutanoyl chloride, dansyl chloride ethylenediamine,
propargyl alcohol, hex-5-yn-1-ol, 1-ethyl-3-(3-dimethylamino-
21,22
PU has been used extensively in biomedical applications
due to its elastic and compliant mechanical properties. propyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS),
However, its hemocompatibility is inadequate, especially for heparin (sodium salt), toluidine blue and Orange Acid II were
use in small diameter blood vessels (#6 mm). To improve its purchased from Aldrich or VWR and used without further
hemocompatibility, a variety of materials such as poly(ethylene purication unless otherwise noted. The silica gel was received
2
3
24
25
glycol) (PEG), phospholipid polymers, sulfobetain, and from Selecto Scientic, Georgia, USA, while the TLC plate was
26
heparin have been integrated onto the PU surface. The most from Analtech, Inc., USA. The PU lm (gauge: 6 mil) was
widely used anticoagulant, heparin, has been introduced onto purchased from American Polylm, Inc.
the polymer surface via either physisorption or covalent
32,33
According to known procedures,
peruorophenyl azide
bonding. The covalent bonding is usually achieved by treating and its derivatives were prepared as depicted in Scheme 2. In
amine furnished polymeric substrates with 1-ethyl-3-(3-dime- brief, substitution of the commercial methyl penta-
thylaminopropyl)carbodiimide/N-hydroxysuccinimide
NHS) activated heparin. However, since EDC/NHS is reportedly acetone reux conditions to give the p-azido counterpart 4.
(EDC/ uorobenzoate with sodium azide was completed under
32
cytotoxic, extensive aer-modication rinsing of the resultant Subsequent hydrolysis under alkaline conditions (NaOH)
2
7
32
materials is necessary. On the other hand, it was reported that afforded the p-azidotetrauorobenzoic acid 3. Then the cor-
carboxyl groups graed onto a PU lm could effectively prolong responding active ester 1 was prepared by coupling 3 with NHS,
28
the partial thromboplastin time (APTT). Therefore, if heparin and subsequently the PFPA-amine 2 was obtained by treating 1
33
is immobilized at the expense of some carboxyl groups, the ratio with excess ethylenediamine. Purication of the synthetic
of carboxyl group/sulfonic group decreases, probably contrib- compounds was performed by ash column chromatography.
uting to the compromise of heparin bioactivity aer its immo- All obtained NMR data were identical to those found in the
bilization. Thus far, the development of simple and effective literature. Azido-compounds, including 2-azido-ethylamine and
heparin immobilization methods via covalent linkage has dansyl-azide 5 (Scheme 2), were prepared according to a pub-
34,35
remained an important goal.
Based on the concept of PFPA chemistry, we proposed to
use light activation to introduce PFPA onto PU for the purpose using a method similar to the synthesis of 5. To dansyl
lished protocol.
13
SYNTHESIS OF DANSYL-ALKYNE (6). Compound 6 was prepared
35
5
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Scheme 1 Schematic illustration of the PU-derived surface photoactive platform (SPP).
Scheme 2 Synthesis of PFPA derivatives and major compounds used in this work.
2 2
chloride (1.04 g, 3.8 mmol) solution in CH Cl (12 mL) and 0.5 Instrumental analysis and characterization
mL pyridine, excess hex-5-yn-1-ol (0.39 g, 4.0 mol) was added.
The resulting solution was stirred for 24 h before being
concentrated under vacuum. The residue was loaded onto a
silica gel column and washed with EtOAc–Hex (1 : 8). The
ATR FT-IR spectra were taken on a Nicolet iS10 spectrometer
(
Thermo Electron Corporation). NMR spectra were recorded at
room temperature (RT) in 5 mm NMR tubes on a Bruker Avance
00 MHz NMR spectrometer. XPS data were acquired with a
3

uorescent fractions were combined to afford 6 as uorescent
Kratos Axis Ultra spectrometer from Kratos Analytical, Inc. The
contact angle was measured by a standard contact angle goni-
ometer (Model 200, with DROPimage standard) from Ram ´e -
Hart Inc. AFM spectra were obtained using a nanoscope
multimedia IIIa atomic force microscope from Veeco Instru-
ments Inc. The images were taken under a tapping mode.
1
oil (1.09 g, 86%); H NMR (CDCl
3
, 300 MHz) d 8.69 (d, J ¼ 8.1 Hz,
H), 8.29–8.34 (m, 2H), 7.56–7.65 (m, 2H), 7.26–7.28 (m, 1H),
.04 (t, J ¼ 6.2 Hz, 2H), 2.95 (s, 6H), 2.06–2.11 (m, 2H), 1.89 (t,
1
4
1
3
J ¼ 2.6 Hz, 1H), 1.71–1.80 (m, 2H), 1.47–1.56 (m, 2H); C NMR
(
CDCl ,75 MHz) d 167.8, 150.8, 131.5, 131.3, 130.9, 130.5, 129.6,
3
123.4, 120.1, 115.9, 83.3, 70.3, 68.9, 45.6, 27.7, 24.2, 17.6.
SYNTHESIS OF PROP-2-YNYL 4-CHLOROBUTANOATE (7). To the
solution of propargyl alcohol (1.0 g, 17.8 mmol, in 15 mL
General procedure for PU surface photo-functionalization
ꢁ1
dichloromethane), triethylamine (1.36 mL, 9.8 mmol) was Methanol solution of 1 or 3 (4.0 mg mL , Scheme 2) was cast
ꢀ
added at 0 C. Subsequently, 4-chlorobutanoyl chloride (1.26 g, on the PU lm and the resulting lm was baked for 20 min in an
ꢀ
8
.9 mmol) was added dropwise over 10 minutes while stirring. oven preheated to 60 C. Then the lm was exposed to UV
Aer stirring at ambient temperature overnight, the solution irradiation (254 nm lamp, 1690 mW cm ) for 10 min at a
was ltered and the solvent removed under reduced pressure. distance of 5 cm. Aer a thorough rinse with methanol, the lm
ꢁ
2
ꢀ
The obtained residue was puried by column chromatography was air dried and stored at 4 C before use (denoted as PU-1 and
1
to give a slightly yellow liquid (1.2 g 85%, H NMR data were PU-3 respectively).
identical to commercial products from Aurora Fine Chemicals
LLC, USA).
The same process was repeated to produce PU-3/PU-1
samples for dye staining. A drop of 3/1 methanol solution was
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spotted on the PU lm; aer photolysis, the lm sample was known amount of aqueous heparin solution (2 mL) was added.
rinsed with ethanol to give samples PU-3-s and PU-1-s respec- The mixture was agitated using a vortex mixer, then n-hexane
ꢁ
1
tively (s ¼ spotting). PU-3-s was then immersed in 0.1 mg mL
(3 mL) was added and the mixture was shaken well to extract
thionin acetate (THA) solution for 8 h, excess dye was washed off the toluidine blue–heparin complex into the organic layer. The
with ethanol, and an image was taken.
toluidine blue remaining in the aqueous phase was thus
determined by absorption at 631 nm. The linear relationship
between the absorbance of residual toluidine blue at 631 nm
and the concentration of heparin in aqueous solution was
obtained as a curve. Next, the amount of immobilized heparin
was determined. Toluidine blue solution (3 mL) was mixed with
an aqueous solution (2 mL), and PU-1-PFPA–heparin (1 ꢂ 1 cm)
was immersed in the mixed solution for 30 min. n-Hexane
Titration of the surface carboxylic acid group on PU-3
The surface carboxylic acid group on PU-3 was determined by
monitoring the absorbance change of THA dye solution before
and aer the electrostatic interaction driven dyeing process
36
using a known procedure. The PU-3 lms were immersed in
ꢁ
1
5
mL of a THA solution in ethanol (concentration 0.1 mg mL )
(3 mL) was then added to ensure uniformity of the treatment.
and the mixture was shaken for 8 h at RT. Then the lms were
removed and rinsed three times. All the washed dye solution
was collected and nally diluted to a constant volume of
The absorbance (631 nm) of the aqueous layers of the solution
was checked and the amount of immobilized heparin was thus
calculated from the calibration curve.
250 mL. The uorescence intensity of the solution was calcu-
lated and the carboxyl group density at the lm surface was
measured by comparing the detected values of the washing Platelet adhesion test
solution with that of the initial dye.
The platelet adhesion test was carried out in a static blood
38
environment according to the literature. Human whole blood
was collected from healthy volunteers and mixed with citrate
solution (10% volume, concentration 3.8%) as an anticoagu-
lant. The blood was centrifuged at 1200 rpm for 10 min at RT to
obtain platelet-rich plasma (PRP). The number of platelets was
determined using a haemocytometer. The platelet concentra-
Graing amines to the PU-1 lm
The PU-1 lm was immersed in an acetonitrile solution of 2-
propynylamine, 2-azido-ethylamine or PFPA-amine 2 (50 mM,
20% ethanol was added in the case of preparing solution 2) and
shaken continuously overnight at RT. Aerward, the lm
5
tion of PRP solution was adjusted to 1 ꢂ 10 cells per mL by
sample was rinsed thoroughly with ethanol, air dried in the
ꢀ
adding phosphate buffered saline (PBS, pH 7.4) to PRP.
PU-1-PFPA–heparin, PU-1-PFPA–heparin-R and untreated PU
dark and stored at 4 C until use. The resulting lms were
named PU-1-alkyne, PU-1-azide, and PU-1-PFPA respectively.
2
samples (1 ꢂ 2 cm ) were equilibrated with PBS (pre-warmed to
ꢀ
3
7 C) for 1 h and then immersed in 1 mL of the separated
Click linkage between PU-1-alkyne and dansyl azide 5
ꢀ
platelet suspension for 2 h at 37 C with 5% CO
2
. Aer incu-
At RT, PU-1-alkyne was immersed in the dansyl azide 5 solution
bation, the samples were taken out and gently washed three
times with PBS to remove the non-adhered platelets. Quanti-
cation of platelet adhesion on the samples was carried out
according to the following equation:
(
0.01 M, t-BuOH–H
2
O ¼ 1 : 1), and Na ascorbate (40% mol) and
Cu (10% mol) were added to initiate the click reaction. Aer
h, the sample was removed, rinsed with water and ethanol,
2
+
2
and nally immersed in ethanol with continuous shaking
overnight to remove the physically adsorbed uorescent dye.
The control sample was produced using the same procedure
platelet adhesion (%) ¼ (N ꢁ N )/N ꢂ 100
t
2
+
except that Cu was not added.
where N is the number of platelets in PRP solution before
contact and N is the number of platelets in PRP solution aer
t
Photo attaching heparin to PU-1-PFPA
contact.
ꢁ
1
Heparin solution (2.5 mg mL , sodium salt) was cast on PU-1-
PFPA and photoreaction was performed in wet conditions for 5 Copper-free click study of PFPA
min. Excess heparin was then removed and the resultant lm
MODEL REACTION. Compound 4 (20 mg, 0.08 mmol) was
was rinsed thoroughly with DI water. The heparin attached lm
PU-1-PFPA–heparin) was air dried in the dark before the static
contact angle measurement, XPS analysis, and platelet adhe-
sion test. The process was repeated using recycled heparin
solution and the obtained lm (PU-1-PFPA–heparin-R, R ¼
recycled) was used to test platelet adhesion properties.
dissolved in propargyl alcohol (0.3 mL, 5.2 mmol) and the
solution was le to stand for 40 min before being checked by
TLC. Then the reaction solution was evaporated at RT and
puried by column chromatography to give a white solid which
(
1
was submitted to H NMR and HRMS analyses. Meanwhile,
another model reaction was performed by dissolving 4 (20 mg,
0
0
.08 mmol) and propargyl alcohol (0.03 mL, 0.52 mmol) in
.4 mL mixed solvent (t-BuOH–H
O ¼ 1 : 1) containing 40% mol
Quantication of immobilized heparin
2
The photo-attachment of heparin was determined by titration Na ascorbate and 10% mol CuSO (calculated based on 4). The
as previously reported. The calibration curve was determined reaction mixture was continuously shaken for 2 h and the
4
37
as follows. To 3 mL toluidine blue solution (stock solution, product was also puried by column chromatography. A proton
25 mg in 0.01 N hydrochloric acid containing 0.2 wt% NaCl), a NMR spectrum of the obtained white solid was then collected.
5
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COPPER-FREE CLICK REACTION ON PU-1-PFPA. PFPA-amine 2 hydrophilicity. The static contact angle of water decreased from
ꢀ
ꢀ
was rst coupled to PU-1-s to give the corresponding PU-1-PFPA- 97.6 (untreated PU) to 81.5 (PU-3) as shown in Fig. 1(a) and (b).
s. Two pieces of PU-1-PFPA-s lms was then immersed in 2,2- The newly introduced carboxylic group makes the lm more
ꢀ
methylbut-3-yn-1-amine overnight at RT and 37 C, respectively. hydrophilic.
36
Next, all the lms were rinsed thoroughly with ethanol before
According to the colorimetric titration method, the surface
ꢁ1
being stained with 0.01 g mL Acid Orange II at pH 3 with carboxylic group density on PU-3 was determined to be 10.3 ꢃ
39
ꢁ2
continuous shaking overnight. The stained sample lms were 1.6 nmol cm . These data are slightly higher compared with
ꢁ
2
rinsed with distilled water followed by 1 mM HCl to remove the carboxylic group density (4.0 nmol cm ) generated on the
40
adsorbed dye molecules before taking the images.
poly(ether imide) surface using a thermochemical reaction.
2
At the same time, PU-1-PFPA (0.5 ꢂ 0.5 cm ) was immersed Atomic force microscopy (AFM) was also performed to evaluate
in 0.5 mL liquid 7 overnight at RT. The obtained lm was then the possible morphological alterations aer the UV irradiation
thoroughly rinsed with ethanol before submitting to XPS anal- process. As shown in Fig. S3,† the untreated PU lm possesses a
ysis. As a control, PU was also treated with 7 by the same smooth surface with a root mean square roughness (R
approach before XPS analysis.
q
) of
8.8 nm, while PU-3 shows a bumpy surface with a higher R
31.5 nm). The increased roughness seems to be mainly caused
q
(
by the surface photoreaction since the control PU lm (which
had gone through the similar treatment as PU-3 except without
Results and discussion
Photograing of p-azidotetrauorobenzoic acid (3) onto PU
the presence of 3) has a R of only 12.5 nm. The fact that model
q
p-azidotetrauorobenzoic acid (3) was synthesized as a model compound 3 is present on a rough surface with a larger surface
compound and photograed on PU to test the photo-coupling area might contribute to the higher surface carboxylic group
feasibility as well as to determine the photograing efficacy. In density.
ꢁ1
ATR spectra (Fig. S1†), the peak 1703 cm (ascribed to the
stretch of C]O in carboxylic acid and ester) intensied aer
photoreaction, suggesting the presence of 3 on the modied PU
Fabrication of PU-1-PFPA as a SPP
(PU-3). Meanwhile, PU-3 was stained with THA, a cationic dye It is usually necessary to confer reactive species both on solid
that can form ionic bonds with carboxylic acid groups via surfaces and on target cargoes for coupling. However, it is
electrostatic interaction. Aer thorough rinsing, a uniform blue challenging to chemically derivatize functional molecules such
color on the modied circular area was visible (Fig. 1(c)). As as carbohydrates. The photosensitive PFPA groups, once
depicted in Fig. 1(e), F 1-s signal in the XPS survey of PU-3 immobilized on solid surfaces, have proven efficient in photo-
41,42
further conrmed the attachment of 3 on PU, since F element capturing carbohydrates with preserved bioactivity.
Simi-
existed only in 3 and not in the untreated PU. The conversion larly, PS simple polymer and PS nano-particles have been photo-
43–45
ratio (c) was calculated to be 53.0% based on XPS data (Fig. S2†), graed on solid surfaces based on PFPA photochemistry.
meaning 3 was graed onto the PU lm at a density of 0.53 Therefore, we rst fabricated PU-1 using an approach similar to
molecule of 3 per PU repeating unit (assuming two nitrogen the fabrication of PU-3 and subsequently coupled 2 onto PU-1 to
atoms in every repeating unit). The introduction of a carboxylic produce the photoactive PU-1-PFPA as depicted in Scheme 1. To
acid group manifested the change of a macroscopic property – validate the introduced phenyl azide group, ATR spectra were
Fig. 1 Characterization of PU-3. Contact angle measurements of PU (a) and PU-3 (b); visualization of THA dye stained PU-3 (c); XPS survey of PU (d) and PU-3 (e).
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Fig. 2 ATR spectra of (a) untreated PU; (b) PU-1; (c) PU-1-PFPA; (d) PU-1-PFPA (back of the film).
collected and are presented in Fig. 2. The spectrum of PU-1-
The heparin density on PU-1-PFPA–heparin was determined
ꢁ1
PFPA (Fig. 2(c)) presented a new peak at 2124.7 cm (ascribed by a colorimetric toluidine blue assay to be 0.64 ꢃ 0.08 mg
ꢁ
2
to –N
was no detectable –N
means that the physically adsorbed 2 could be removed by and a silicone surface (0.68 mg cm ). Usually higher heparin
3
of PFPA) as compared with that of PU-1 (Fig. 2(b)). There cm , which is comparable to that achieved on other
ꢁ
2
46
3
peak on the back of PU-1-PFPA, which substrates such as a polysulfone membrane (0.86 mg cm )
ꢁ
2
47
ꢁ
1
rinsing. At the same time, another peak (1654.44 cm ) was density can be achieved by adopting polymeric spacers, such as
ꢁ
2
27
observed, which corresponds to the amide bond that was newly chitosan (1.37–2.97 mg cm
formed in PU-1-PFPA. The differences between the ATR spectra cm ). Polymeric spacers provide a benecial reaction
of PU-1, PU-1-PFPA and PU-1-PFPA (back) demonstrated that 2 microenvironment with less steric hindrance and more
)
and polyallylamine (3.6–4.1 mg
ꢁ
2
48
was chemically conferred onto PU-1.
accessible bonding sites for heparin coupling. In comparison
with the PU substrate graed with chitosan spacer and heparin
27
(PU–chitosan–heparin),
PU-1-PFPA–heparin achieved
a
Functionalization of PU-1-PFPA with heparin
similar platelet adhesion reduction level (from 43.1 ꢃ 2.2% to
On the PU-derived SPP (PU-1-PFPA), heparin was covalently 17.6 ꢃ 0.8% vs. from 27% to 13%) with only half the surface
ꢁ2
ꢁ2
attached via the photo-coupling method. Aer a thorough rinse, heparin concentration (0.64 ꢃ 0.08 mg cm vs. 1.37 mg cm ).
the resultant sample PU-1-PFPA–heparin was air dried over- This is probably due to the fact that the photo-coupling
night before the measurement of the static contact angle. The method to immobilize heparin is quite different from the
ꢀ
contact angle distinctly decreased from 90.0 ꢃ 0.7 (PU-1-PFPA) coupling methodology based on carbodiimide chemistry. In
ꢀ
27,46,49
to 78.3 ꢃ 0.3 (PU-1-PFPA–heparin) aer the attachment of the latter case, excess EDC (70 equiv.) is usually used
to
hydrophilic heparin. To further conrm the attachment, PU-1- ensure the coupling efficacy. Given that stoichiometry, all the
PFPA–heparin was subjected to XPS analysis and the S 2p signal carboxylic groups on the heparin chain are activated. As a
was detected (Fig. 3). Since S uniquely exists in heparin, the result, when the polysaccharide chain with multiple activated
observed S 2p signal suggests the successful attachment of binding sites is exposed to surface amines, more than one
heparin on PU-1-PFPA. Meanwhile, the F atomic concentration activated carboxylic acid groups in the same heparin chain
decreased signicantly from 3.40% to 0.68% (Fig. 3), which is may couple to the polymer surface, leading to multipoint
reasonable considering that once PU-1-PFPA is covered with attachment and relatively rigid heparin chains (Fig. S4†). For
heparin (F-free polymer) as a result of photolysis, many of the F the photo-coupling method, when the hydrophobic surface of
ꢀ
atoms of PFPA might be buried to a depth undetectable by XPS. PU-1-PFPA (contact angle 90.0 ꢃ 0.7 ) contacts the heparin
As the rst event of blood clotting cascades, platelet adhe- solution, it is likely that very small amounts of heparin mole-
sion has been the accepted evaluator for the hemocompatibility cules lay themselves completely on the PU-1-PFPA surface so
of blood contacting medical devices. As shown in Fig. 4, the that few heparin chains are captured via multi N–C bonds
percentage of platelet adhesion on the untreated PU lm (Fig. S4†). The photo-coupling method under wet conditions
reached 43.1 ꢃ 2.2%, a high level of coagulation. Aer the probably produced free polysaccharide chains that seemed to
attachment of heparin, the platelet adhesion reduced signi- stand on the PU-1-PFPA surface, supporting the fact that
cantly to 17.6 ꢃ 0.8%, indicating that PU-1-PFPA–heparin matchable platelet adhesion reduction was achieved despite a
effectively prevented platelet aggregation and adsorption.
reduced heparin density.
5
76 | J. Mater. Chem. B, 2013, 1, 571–582
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Journal of Materials Chemistry B
Fig. 3 XPS survey of (a) PU-1-PFPA and (b) PU-1-PFPA–heparin.
to produce the modied lm PU-1-PFPA–heparin-R (“R” refers
to recycled).
Interestingly, the percentage of platelet adhesion on the lm
immobilized with recycled heparin – PU-1-PFPA-heparin-R –
was only 18.9 ꢃ 2.8%, which is not signicantly different from
that on PU-1-PFPA-heparin (17.6 ꢃ 0.8%, Fig. 4). By comparing
the weight of recycled heparin solution to that of the initially
casted heparin solution, we estimate the recovery to be no less
than 94%. This indicates that the PFPA photo-coupling strategy
for heparin graing is more economical than the EDC/NHS
coupling method where excess heparin with all carboxylic acid
groups pre-activated is usually required for heterogeneous
immobilization on a solid surface.
Fig. 4 Quantification of platelet adhesion on untreated PU, PU-1-PFPA–heparin
and PU-1-PFPA–heparin-R (R ¼ recycled). The diagram includes t-test results (n ¼
3
) with respect to untreated PU (*p < 0.05, **p < 0.01 and ***p < 0.001).
The rst advantage of PU-derived SPP is that a great variety of
molecules, regardless of the molecular structure or architecture,
could be immobilized by this simple and fast photo-activation
The photo-attaching process was performed in wet condi- procedure as long as they can endure UV irradiation for a few
tions, and only those heparin molecules in covalent bonding minutes. Second, the photo-graing efficacy can be conve-
13
distance with the surface generated nitrenes could be niently controlled simply by adjusting the light intensity.
captured, leaving the remaining heparin molecules intact Spatial specic immobilization is also possible using photo-
15
in the aqueous solution. Hence, it might be possible to masks during UV irradiation. Third, since phenyl azide deriv-
recycle the vast majority of heparin in the solution, and retain atives have been photo-immobilized onto various substrate
5
0
51
the antithrombotic activity. To test this hypothesis, the materials such as polyimide, polystyrene polypropylene, and
15
heparin solution that had been exposed to UV irradiation even inorganic carbon materials, this two step general modi-
during the photograing process was salvaged (by pipette cation method (PU / PU-1 / PU-1-PFPA) to produce SPP
tips) and again cast on PU-1-PFPA for another graing process could readily be extended to these substrate materials.
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Fabrication of a surface clickable platform (SCP)
validate the surface alkynyl groups, we next integrated a
35
dansyl-azide 5 onto PU-1-alkyne via the CuAAC chemistry
method. Aer thorough rinsing, a uniform green uorescence
was observed (Fig. 5(d)), while only blue autouorescence was
seen on PU-1-alkyne (Fig. 5(c), the control without the addi-
As discussed above, the SPP of PU-1-PFPA is capable of directly
capturing various molecules upon UV irradiation, but the
methodology is not applicable to those light sensitive func-
tionalities that need to be conferred on the surface. As such,
universal platforms that could achieve the attachment of func-
tional moieties via the robust and versatile linkage methodology
such as the copper-catalyzed azide–alkyne cycloaddition
2
+
tion of Cu ). This result indicates the successful formation
of uniformly distributed alkynyl groups on the surface of
PU-1-alkyne.
With the established SCP of PU-1-alkyne/PU-1-azide, a variety
of functional reagents containing bioorthogonal tags could be
conveniently conferred via the CuAAC chemistry method. To
this end, PU-1-azide/PU-1-alkyne could be regarded as a mutual
supplement to PU-1-PFPA in that the former provides a robust
and versatile bonding method to immobilize alkynyl/azido
cargoes with light-sensitive functionalities, while the latter is
capable of readily integrating various light-insensitive func-
tionalities without any chemical structural modication.
(
CuAAC, also named “click” reaction) are also desirable. In
principle, direct photo-coupling of PFPA–alkyne or PFPA–azide
derivatives onto the PU surface is the most effective modica-
tion method. However, the PFPA–alkyne derivative can undergo
a self-click reaction even during purication which is prob-
lematic. On the other hand, alkyl azides could also undergo
photodecomposition when exposed to UV irradiation. Hence
we herein attached 2-azidoethylamine and 2-propynylamine on
PU-1 via a two-step method to produce SCP as PU-1-azide and
PU-1-alkyne, respectively (Fig. 5).
52
53
Copper-free click performance of PFPA
The ATR spectrum of PU-1-azide presented an expected
ꢁ
1
55
azide peak (2102.15 cm ) (Fig. S5†), suggesting the success- CuAAC has found numerous applications in drug design,
56
57
ful covalent attachment of 2-azidoethylamine on PU-1. chemical biology and materials science, but entails the use of
ꢁ
1
The azide peak shied from 2124.7 cm
2
(PU-1-PFPA) to a copper salt catalyst, which exhibits in vivo toxicities in bio-
ꢁ
1
58
102.2 cm
(PU-1-azide). The higher wavenumber of the logical system applications. To this end, the strain-promoted
azide peak in PU-1-PFPA is probably due to the electron azide–alkyne cycloadditions (SPAAC) have been developed as a
withdrawing effect of the peruorophenyl group. As presented copper-free alternative with suitable reactivity for bioorthogonal
59,60
in Fig. 5(a) and (b), high-resolution XPS N 1s spectrum was chemistry applications.
However, the preparation of
curve-tted and a distinct N 1s peak was resolved at a binding strained cycloalkynes usually requires extensive and laborious
energy of 404.5 eV, which is attributed to the central nitrogen synthetic work, so this method is rarely appropriate for
of the azide moiety. The other two nitrogen atoms of the azide straightforward synthetic applications. Therefore, the develop-
5
4
moiety possess lower binding energies and the correspond- ment of a simple version of copper-free cycloaddition reaction is
ing peaks are consequently hidden in the main peak of amide highly appreciated.
nitrogen (400.4 eV). The satellite N 1s peak conrms the
To our knowledge, the rst copper-free cycloaddition
successful attachment of 2-azidoethylamine on PU-1. To between PFPA and alkynes was mentioned by Yan and
Fig. 5 Formation and characterization of a surface clickable platform (SCC). High-resolution XPS N 1s spectra of PU-1-azide (a) and PU-1 (b); visualization of PU-1-
alkyne (c) and PU-1-alkyne-5 (d).
5
78 | J. Mater. Chem. B, 2013, 1, 571–582
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coworkers, but no further details have been presented. We
Journal of Materials Chemistry B
52
As subsidiary evidence, 2-azidoethyl benzoate, the synthetic
61
initially attempted to prepare prop-2-ynyl 4-azido-2,3,5,6-tetra- alkyl–azide, was dissolved in propargyl alcohol at RT, but no
uorobenzoate (8) by esterication of 3 with propargyl alcohol visible reaction was observed on the TLC plate within 40 min,
Fig. S6†). The derivative 8 contains the shortest linker –CH –, further suggesting that the copper-free click reaction between 4

(
2
probably decreasing the crosslinking possibility during the and propargyl alcohol is specically due to the presence of the
photo-coupling process. However, self-click reactions pro- PFPA moiety.
ceeded in the purication process (see Fig. S6†). Based on the
Subsequently, the feasibility of copper-free click reaction on
observation on thin layer chromatography (TLC), we could draw PU-1-PFPA was tested. Initially, PU-1-PFPA-s (s ¼ spotting) was
a tentative conclusion that the self-click reaction is concentra- fabricated to be treated with propiolic acid to confer carboxylic
tion dependent and thermally promoted.
acid groups that could be stained by THA. However, PU-1-PFPA
We then investigated the similar potential copper-free click was found to be readily dissolvable in propiolic acid. We then
behaviour of PFPA groups on PU-1-PFPA. First, a model reac- turned to 2,2-methylbut-3-yn-1-amine, a liquid alkyne-amine, in
tion was performed in an open centrifuge tube by mixing which PU-1-PFPA-s was immersed overnight in a dark place (at
ꢁ
1
ꢁ1
ꢀ
diluted solutions of 4 (0.15 mol L ) and 6 (0.15 mol L ) at RT and 37 C respectively). Aer being stained with Acid Orange
ꢀ
RT and 37 C, respectively. The reaction process was moni- II, the casting region (PU-1-PFPA) for both samples exhibited a
tored by TLC. No new visible spot was observed (data not distinctly deeper brownish color (Fig. 7(a) and (b)) in contrast to
shown) on the TLC plates for both samples within 40 min; the unspotted area on the lms. No drastic difference was
ꢀ
however, a new spot appeared on the baseline (Fig. S6†) aer observed between the samples clicked at RT (Fig. 7(a)) and 37 C
the solvent evaporated overnight. The new spot that formed at (Fig. 7(b)). This observation indicates that the copper-free
ꢀ
3
7
C seemed intensied in comparison with the spot cycloaddition between PU-1-PFPA and 2,2-methylbut-3-yn-1-
produced at RT. These observations indicate that the copper- amine proceeded smoothly in the absence of the copper catalyst
free click between 4 and 6 is concentration dependent and at RT. To further conrm this spontaneous click reaction, PU-1-
thermo-promoted, which is in accord with the self-click of 8. PFPA was treated with 7, a liquid alkyne containing chlorine
To further test this copper-free click hypothesis, we next atoms. As expected, a Cl 2p signal was detected in the XPS
directly dissolved 4 in propargyl alcohol to nd that a new spectra (Fig. 7(c) and (d)) while no Cl 2p peak was observed on
spot was observed on the TLC plate within 40 min (Fig. 6). The the back of the resultant lm (PU-1-PFPA-7, Fig. 7(e)) and the
reaction mixture was submitted to HRMS analysis and a new control sample (PU, data not shown). Meanwhile, the high-
+
peak with [M + H] ¼ 306.0511 (calculated 306.0502) was resolution N 1s spectra were curve-tted and resolved at the
detected, which corresponded to the newly produced triazole binding energy of 401.7 eV and 400.5 eV with a ratio of 1 : 2
adduct. Meanwhile, the CuAAC reaction between 4 and (Fig. 7(f) and (g)), which are attributed to the nitrogen atoms of
1
35
propargyl alcohol was also performed at RT. The H NMR the newly formed triazole adduct. All the evidence collectively
spectra of the puried product 9 and copper-free click adduct supports the feasibility of copper-free click of alkynes onto the
(
isomer mixture) were collected. Based on the analysis of tri- surface of PU-1-PFPA.
azole-H integration, it could be concluded that the copper-free Metal free cycloaddition of azide with alkynes usually
click reaction between 4 and propargyl alcohol afforded a remains sluggish due to the relatively high kinetic-energy barrier
ꢁ
1
62
mixture of 1,4- and 1,5-regioisomers with a ratio of ca. 4 : 1 (ca. 26 kcal mol ), but the reaction could be promoted
0.999 : 0.249) (Fig. 6). by prolonged heating, resulting in a mixture of 1,4- and
(
Fig. 6 Model study of copper-free click reactions between the PFPA methyl 4-azido-2,3,5,6-tetrafluorobenzoate (compound 4) and the alkyne prop-2-yn-1-ol.
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Fig. 7 Study of copper-free click reactions on PU-1-PFPA. Acid Orange II staining of PU-1-PFPA samples copper-free clicked with 2,2-methylbut-3-yn-1-amine at (a) RT
ꢀ
ꢀ
and (b) 37 C respectively; (c–e) high-resolution XPS Cl 2P spectra of PU-1-PFPA-7 (c and d: spectra for the films on which copper-free click reactions proceeded at 37
C
ꢀ
ꢀ
and RT, respectively) and its back (e, 37 C); (f–g) high-resolution XPS N 1s spectra of PU-1-PFPA-7 films obtained from copper-free click reactions proceeded at 37 C and
RT, respectively.
1,5-regioisomers. In contrast, copper(I) catalysis dramatically oriented immobilization of alkynyl functional moieties.
accelerated the click reaction between azides and terminal Without any chemical derivatization, heparin was photo-
63
alkynes to give excellent yields of 1,2,3-triazole. An alternative coupled to PU-1-PFPA with well preserved bioactivity. Heparin
to CuAAC is SPAAC, which possesses low activation energy (DE ¼ that remained ungraed during the photocoupling process
ꢁ
1
8
.0 kcal mol ) compared to the strain free alkynes cycloaddi- could be recovered and reused for another photo-attachment
ꢁ1
64
65
tion (DE ¼ 16.2 kcal mol ). Ni and coworkers reported a experiment on PU-1-PFPA to achieve the same level of antico-
copper-free click reaction between PFPA and phenyl alkynes in agulant activity as that of freshly prepared heparin solution.
the crystalline phase due to the optimal spatial arrangement This work manifests the idea of combining photo-anchoring
of the phenyl alkyne and the tetrauorophenyl azide moiety, but and capturing strategies based on PFPA chemistry to covalently
the cycloaddition reaction under this condition proceeds slowly immobilize functional moieties, especially those that cannot be
and requires 14 days for satisfactory conversion. Zhang and readily derivatized, onto chemically inert thermoplastic poly-
coworkers found that uorinated benzenes are more aromatic meric substrates.
than benzene in that the p
z
lone pair electrons of F atom
Meanwhile, it has been found that surface immobilized
66
participate in the formation of hexagon p current. On the other PFPA can undergo copper-free cycloaddition with alkynes in the
hand, due to the strong electronegativity of the F atom, the interface even at RT, serving as a convenient and biologically
uorinated benzene moiety of PFPA is a more electron-decient benign conjugate methodology for achieving oriented graing
structure. We speculated that the increased aromaticity and the of bioactive reagents. In summary, the photoactive and copper-
electron deciency probably decreased the activation energy for free clickable surface developed on the basis of PFPA photo-
PFPA to achieve the transition state for feasible cycloaddition, coupling and click chemistry exemplies facile tuning of the
leading to an obvious self-click phenomenon between PFPA and surface chemistry of chemically inert thermoplastic polymers
alkynes. The detailed kinetic data and the optimal reaction for biomedical applications.
conditions for the copper-free cycloaddition between PFPA and
alkynes will be investigated in other reports.
Acknowledgements
This work was supported by the Natural Sciences and Engi-
Conclusions
neering Research Council of Canada (NSERC) Discovery grant
Using a two-step modication process based on PFPA photo- (Grant no: RGPIN/372048-2009), the Manitoba Health Research
coupling chemistry, we have successfully constructed a versatile Council (MHRC) Establishment grant, and Collaborative Health
and exible surface platform PU-1-PFPA for both the immobi- Research Projects (CHRP) operating grant (Grant no: CHRP
lization of bioactive agents lacking reactive groups and the 413713-2012).
5
80 | J. Mater. Chem. B, 2013, 1, 571–582
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Journal of Materials Chemistry B
2
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