Post-assembly derivatization of electrospun nanofibers via strain-promoted azide alkyne cycloaddition

Article abstract of DOI:10.1021/ja307647x

A primary amine-derivatized 4-dibenzocyclooctynol (DIBO) was used to initiate the ring-opening polymerization of poly(γ-benzyl-l-glutamate) (DIBO-PBLG). This initiator yields well-defined PBLG polymers functionalized with DIBO at the chain termini. The DI

Full text of DOI:10.1021/ja307647x

Article
pubs.acs.org/JACS
Post-Assembly Derivatization of Electrospun Nanofibers via Strain-
Promoted Azide Alkyne Cycloaddition
Jukuan Zheng,† Kaiyi Liu,† Darrell H. Reneker,† and Matthew L. Becker*,†,‡
†Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States
‡Center for Biomaterials in Medicine, Austen Bioinnovation Institute in Akron, Akron, Ohio 44308, United States
S
* Supporting Information
ABSTRACT: A primary amine-derivatized 4-dibenzocyclooc-
tynol (DIBO) was used to initiate the ring-opening polymer-
ization of poly(γ-benzyl-L-glutamate) (DIBO-PBLG). This
initiator yields well-defined PBLG polymers functionalized
with DIBO at the chain termini. The DIBO end group further
survives an electrospinning process that yields nanofibers that
were then derivatized post-assembly with azide-functionalized
gold nanoparticles. The availability of DIBO on the surface of
the fibers is substantiated by fluorescence, SEM, and TEM
measurements. Post-assembly functionalization of nanofiber
constructs with bioactive groups can be facilitated easily using this process.
olymeric nanofibers are applied increasingly to regener-
glutamate-N-carboxyanhydride (Bz-L-GluNCA) to yield a 4-
Pative medicine applications due to dimensional similarities dibenzocyclooctynol functionalized poly(γ-benzyl-L-glutamate)
with collagen fibers in the natural extracellular matrix.1
Nanofibers are fabricated using melt- or electrospinning
(DIBO-PBLG). PBLG is a versatile, degradable material that
can adopt α-helix and β-sheet conformations17 and is being
investigated for cell adhesion and proliferation when used with
protein preadsorption techniques.18,19 The high binding affinity
of calcium to PBLG is also promising for bone regeneration
applications.20
processes that are able to control both size and morphology
using a variety of conditions including solvent, concentration,
and additives.2 Many of the reported fiber technologies have
focused on using polymeric materials, which are utilized in
FDA-approved applications or devices. While useful, these
materials in general lack specific functionality that would guide
or direct specific biological functions.3 For regenerative
medicine, fabrication of biomolecule-derivatized nanofibers
that enhance cell−material interactions is important.4 The
derivitization of nanofibers often requires multiple procedures,
including plasma treatment, wet chemical methods, surface
graft polymerization, and co-electrospinning of surface active
agents with polymers.3 Each of these modifications are time-
and resource-intensive to optimize and may lead to immune
specific reactions and biocompatibility problems.5 Recently,
click chemistry,6,7 especially strain-promoted azide-alkyne
cycloaddition (SPAAC) reactions, has emerged as a promising
biofunctionalization method due to its high efficiency,
orthogonality, and tolerance of reaction conditions in the
presence of cellular systems.8 SPAAC has been investigated in
biological hydrogels,9−11 polymer side-chain functionaliza-
tion,12 and as a labeling method for biological systems,13,14
among others. Several generations of cyclooctynes have been
developed recently by both the Boons and Bertozzi groups.15,16
To our knowledge, amine-derivatized cyclooctynes have not
been utilized previously as an initiator for ring-opening
polymerization. In this work, 4-dibenzocyclooctynol (DIBO)
functionalized with a primary amine group was used as an
initiator for the ring-opening polymerization of γ-benzyl-L-
The DIBO-PBLG was synthesized as described in Scheme 1.
The strained DIBO precursor was synthesized according to
previously described methods.15,21 The DIBO was derivatized
with p-nitrophenyl chloroformate and further reacted with
excess hexamethylene diamine to yield the primary amine-
derivatized DIBO compound, 6. Bz-L-GluNCA was synthesized
and purified by flash chromatography according to previously
reported procedures.22,23
1
The H NMR spectrum of the DIBO initiator and DIBO-
PBLG are shown in Figure 1. The initiator was used
immediately after purification. In a series of polymerizations
the amine-derivatized DIBO was used as an initiator for the
ring-opening polymerization of Bz-L-GluNCA in anhydrous
DMF under nitrogen for 3 days to yield DIBO-functionalized
PBLG. The molecular weight of DIBO-PBLG increased linearly
with increasing feed ratio from 50:1 to 500:1. The
corresponding molecular weight distribution increased from
1.14 to 1.29, which demonstrated that these polymerizations
are well controlled and exhibit linear growth kinetics with
increasing feed ratio. The various feed ratio conditions and the
resulting molecular weights and molecular weight distributions
of the resulting polymers were measured via DMF phase SEC
Received: August 2, 2012
© XXXX American Chemical Society
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Scheme 1. Synthesis of Dibenzocyclooctynol Functionalized
Poly(γ-benzyl-L-glutamate) (DIBO-PBLG)
Figure 2. SEC results of DIBO-PBLG with different feed ratios. SEC
was run in DMF with LiBr (0.1 mol/L) under 50 °C.
collection of electrospun fibers. Samples were silver coated with
an SPI sputter coater before the SEM observation. From SEM
micrographs, we obtained fibers with diameters near 1 μm in
diameter (Figure 3).
1
Figure 1. H NMR spectrum of the DIBO-derivatized initiator and
DIBO-functionalized PBLG in DMSO-d6.
Figure 3. SEM pictures of nanofibers from electrospinning show
highly uniform fiber diameters.
and are shown in Figure 2. The high molecular mass shoulders
that appear at increasing feed ratios are consistent with what
has been reported by others using amine -based initiators for
the polymerization of PBLG and are most likely due to water-
initiated polymer.24,25
To confirm the survival of the strained DIBO group
following the electrospinning process, the fibers were dissolved
in DMF, and UV−vis spectra of the solutions showed the
presence of optical transitions near 306 nm, which correspond
to the alkyne group in DIBO (Figure 4), before and after the
fabrication process. With the survival of DIBO group through
electrospinning, the availability of DIBO on the surface of fibers
for biofunctionalization was probed.
The extent of DIBO available on the surface of the
nanofibers to react was quantified using UV−vis spectroscopy.
A solution of 9-methyleneazidoanthracene in methanol, which
is a non-solvent for PBLG, was allowed to react with a mat of
electrospun fibers. Concurrently, a solution of 9-methylenea-
zidoanthracene in N,N-dimethyl formamide, which is a good
solvent for PBLG, was allowed to react with an equivalent
solution of the DIBO-initiated PBLG. The absorbance of the
nanofiber post-functionalization and PBLG-DIBO with identi-
cal concentrations was measured following the reaction, and
from the reduction in absorbance of the alkyne transition in
The DIBO-derivatized PBLG was then used as a functional
precursor for electrospinning of nanofibers to generate a
copper-free clickable scaffold. DIBO-PBLG (Mn = 128 K, PDI
= 1.29) and the unmodified PBLG (Mw 150,000−350,000,
Sigma) were both prepared in a 12 wt % 1,4-dioxane solution.
Each polymer solution was held in a glass pipet and was
electrospun from the orifice having an inner diameter of 300
μm at the tip. The electric potential was 6 kV over a 22 cm tip-
to-collector distance for the modified polymer and 12 kV over a
27 cm tip-to-collector distance for the unmodified polymer. A
proper positive air pressure was applied on the surface of the
solution to maintain the feeding rate. Fibers were collected on
conductive glass slides for the subsequent fluorescence
measurements, silicon wafers for SEM observation, and on
copper grids for TEM observation. Each type of collector was
placed on top of a large grounded aluminum foil mat for the
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nanoparticles. Fiber-loaded silicon wafers were immersed in a
solution of azide-functionalized gold nanoparticles (50 nm,
Nanocs, diluted 500 times) for 5 h, after which the wafer was
washed with nanopure water (18 MΩ/cm−1) and dried under
vacuum. The SEM images clearly showed the presence of gold
nanoparticles on the surface of the fibers, which confirmed that
the DIBO group on the surface of fibers are available for
functionalization (Figure 6 A,B). A control experiment was
Figure 4. UV spectra of DIBO-PBLG before and after electrospinning
indicate that the strained cyclooctyne survives the scaffold fabrication
process (top). A second experiment measuring the residual DIBO left
following a cycloaddition reaction, which indicated approximately 26%
of the groups were able to react (bottom).
Figure 6. SEM micrographs of gold nanoparticle (50 nm) function-
alized fibers (A, B) and unmodified fibers as control (C, D). The scale
bar for images A, B, C, and D is 600, 100, 300, and 200 nm,
respectively.
DIBO, the fraction of the DIBO group on the surface of fiber
that is available for derivatization is 26 5%.
performed using fibers composed of unmodified PBLG, which
showed conclusively that the nonspecific physical adsorption of
gold nanoparticles to the surface of fibers is negligible (Figure 6
C,D).
A post-assembly experiment utilizing an azide-containing
fluorescence probe (Chremo 488 azide) was conducted. A glass
coverslip containing fibers was immersed in a 0.4% (mg/mL)
solution of fluorescence probe for 5 min. Following the brief
incubation, the fiber-loaded coverslip was removed, washed
with methanol, and dried under nitrogen. Using fluorescence
microscopy, we clearly showed that the azide-functionalized
fluorescence probe reacted with the DIBO group on the surface
of fibers. Underivatized PBLG exhibited no fluorescence
following the methanol wash (Figure 5). These experiments
indicate the availability of the DIBO groups on the surface for
functionalization through strain-promoted azide-alkyne cyclo-
addition.
Additional experiments with transmission electron micros-
copy (TEM) confirmed the existence and availability of DIBO
groups on the surface of the fibers. TEM grids loaded with
nanofibers were immersed in an azide-functionalized gold
nanoparticle solution for 1 h at ambient temperature, after
which the grid was washed with nanopure water (18 MΩ/
cm−1) and dried under vacuum. TEM images showed that gold
nanoparticles are present on the surface of the nanofibers.
Control experiments with unmodified nanofibers showed that
no nonspecific physical adsorption of gold nanoparticles was
evident following immersion in the nanoparticle containing
solution (Figure 7). Due to the relatively large size of
nanofibers (chosen for visualization purposes), only small
portions of the fibers are shown in the SEM and TEM
micrographs.
Further proof of the existence of DIBO group on the surface
of the fibers was obtained from SEM micrographs of the fibers
following immersion in a solution of azide-functionalized gold
In conclusion, we demonstrate the utility of an amine-
derivatized DIBO unit as an initiator for ring-opening
polymerization for the first time. Additionally we show that
the DIBO group survives an electrospinning procedure. The
resulting nanofiber scaffold is then available for post-assembly
functionalization with any number of azide-derivatized
molecules. The availability of copper-free click-chemistry-
based biofunctionalization sites on the surface of nanofibers
offers versatile approach to create highly functional scaffolds,
which we feel will be useful in a variety of promising
applications in regenerative medicine.
Figure 5. Fluorescence images of ChremoTM azide stained fibers (A)
and an optical image of unmodified fibers (B).
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ASSOCIATED CONTENT
* Supporting Information
Synthetic details and characterization of the DIBO and amine-
derivatized DIBO. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
becker@akron.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge financial support from the
Akron Functional Materials Center and the Austen Bioinno-
vation Institute in Akron.
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