Clickable degradable aliphatic polyesters via copolymerization with alkyne epoxy esters: Synthesis and postfunctionalization with organic dyes

Article abstract of DOI:10.1021/ja505629w

Degradable aliphatic polyesters are the cornerstones of nanoparticle (NP)-based therapeutics. In this paradigm, covalent modification of the NP with cell-targeting motifs and dyes can aid in guiding the NP to its destination and gaining visual confirmation. Therefore, strategies to impart chemistries along the polymer backbone that are amenable to easy modification, such as 1,3-dipolar cycloaddition of an azide to an alkyne (the click reaction ), could be significant. Here we present a simple and efficient way to introduce alkyne groups at high density in aliphatic polyesters without compromising their crystallinity via the copolymerization of cyclic lactones with propargyl 3-methylpentenoate oxide (PMPO). Copolymers of lactic acid and ε-caprolactone with PMPO were synthesized with up to 9 mol % alkyne content, and accessibility of the alkyne groups to the click reaction was demonstrated using several dyes commonly employed in fluorescence microscopy and imaging (Cy3, ATTO-740, and coumarin 343). In order to establish the suitability of these copolymers as nanocarriers, copolymers were formulated into NPs, and cytocompatibility, cellular uptake, and visualization studies undertaken in HeLa cells. Dye-modified NPs exhibited no quenching, remained stable in solution for at least 10 days, showed no cytotoxicity, and were readily taken up by HeLa cells. Furthermore, in addition to enabling the incorporation of multiple fluorophores within the same NP through blending of individual dye-modified copolymers, dye-modified polyesters offer advantages over physical entrapment of dye, including improved signal to noise ratio and localization of the fluorescence signal within cells, and possess the necessary prerequisites for drug delivery and imaging.

Full text of DOI:10.1021/ja505629w

Article
pubs.acs.org/JACS
Clickable Degradable Aliphatic Polyesters via Copolymerization with
Alkyne Epoxy Esters: Synthesis and Postfunctionalization with
Organic Dyes
Nele S. Teske,† Julia Voigt,† and V. Prasad Shastri*,†,‡
†Institute for Macromolecular Chemistry, Hermann Staudinger Haus, University of Freiburg, Stefan-Meier Str. 31, 79104 Freiburg,
Germany
‡BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schanzlestr. 18, 79104 Freiburg, Germany
̈
S
* Supporting Information
ABSTRACT: Degradable aliphatic polyesters are the cornerstones
of nanoparticle (NP)-based therapeutics. In this paradigm, covalent
modification of the NP with cell-targeting motifs and dyes can aid in
guiding the NP to its destination and gaining visual confirmation.
Therefore, strategies to impart chemistries along the polymer
backbone that are amenable to easy modification, such as 1,3-dipolar
cycloaddition of an azide to an alkyne (the “click reaction”), could
be significant. Here we present a simple and efficient way to
introduce alkyne groups at high density in aliphatic polyesters without compromising their crystallinity via the copolymerization
of cyclic lactones with propargyl 3-methylpentenoate oxide (PMPO). Copolymers of lactic acid and ε-caprolactone with PMPO
were synthesized with up to 9 mol % alkyne content, and accessibility of the alkyne groups to the click reaction was demonstrated
using several dyes commonly employed in fluorescence microscopy and imaging (Cy3, ATTO-740, and coumarin 343). In order
to establish the suitability of these copolymers as nanocarriers, copolymers were formulated into NPs, and cytocompatibility,
cellular uptake, and visualization studies undertaken in HeLa cells. Dye-modified NPs exhibited no quenching, remained stable in
solution for at least 10 days, showed no cytotoxicity, and were readily taken up by HeLa cells. Furthermore, in addition to
enabling the incorporation of multiple fluorophores within the same NP through blending of individual dye-modified
copolymers, dye-modified polyesters offer advantages over physical entrapment of dye, including improved signal to noise ratio
and localization of the fluorescence signal within cells, and possess the necessary prerequisites for drug delivery and imaging.
as solvents can swell the polymer matrix or destabilize the
nanoparticle suspension and compromise end use. While the
synthesis of functionalized lactones is time-consuming and
often plagued with low yields, methods that are much easier to
implement have been reported, including the chain-end
modification route14 and direct functionalization of the polymer
backbone.17−19 Nevertheless, systems that combine ease of
synthesis with simple predictable coupling chemistries can
enhance the application space of these polyesters.
With regard to functional groups that might be considered
for further biofunctionalization, the “click reaction” involving
1,3-dipolar cycloaddition of azides with alkyne groups features
prominently,20 as it is an atom-economical synthetic route that
can be quantitative under the right conditions. Bertozzi and co-
workers demonstrated that with strained cyclooctynes, 1,3-
dipolar cycloaddition in almost quantitative yields without the
need for copper(II) catalysts can be achieved.21 There are
several reports in the literature on the synthesis of aliphatic
polyesters bearing azide or alkyne groups via copolymerization
of derivatized lactones.22−27 Alkyne groups provide unique
INTRODUCTION
■
Aliphatic polyesters derived from cyclic lactones [ε-caprolac-
tone (εCL), lactide and glycolide] represent the major family of
polymers currently used in medical applications. These
applications range from implantable and injectable drug
delivery systems1−4 to support structures (porous scaffolds)
for cell seeding and cultivation to coatings for medical
devices.5−10 In recent years, copolymers of lactide and glycolide
have seen extensive use in the development of injectable
nanoparticles (NPs) for targeted therapy and imaging.11,12 To
increase the utility of degradable polyesters for targeted
therapeutics, much effort has focused on developing monomers
bearing chemistries suitable for functionalization with homing
peptides, antibodies, and/or imaging agents. Two approaches
have been explored to date: (1) synthesis of lactone derivatives
possessing functional groups that are amenable to postfunction-
alization13−15 and (2) direct functionalization of the nano-
particle, device, or scaffold surface by combining for example
controlled hydrolysis of the surface with carbodiimide coupling
of the desired chemical entity.16 Although the latter approach
ensures that the desired information is indeed on the surface of
the nanoparticle, this methodology for modification may be
incompatible with the application or chemistry of the polymer,
Received: June 12, 2014
© XXXX American Chemical Society
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Scheme 1. General Scheme for the Synthesis of Alkyne Epoxy Esters and Alkyne-Functionalized Aliphatic Polyesters
access to simple chemistry to introduce dyes and contrast
agents for imaging applications. However, in order to benefit
from the simplicity of click chemistry, systems that can be easily
realized is the key. While most lactone synthesis involves
complex synthetic methodology,22 the work by Emrick and co-
workers26,27 stands out as it employs the copolymerization of α-
propargyl-δ-valerolactone, which is easily accessible, to
synthesize alkyne-modified poly(ε-caprolactone) (pCL). How-
ever, the synthesis of other alkyne-bearing variants of
valerolactone, such as those possessing degradable linkers, is
not trivial. In a recent publication, we reported the
functionalization of polyesters using α,ω-epoxy esters.28,29
This approach offers two advantages over other published
methods: (1) since epoxides can undergo ring-opening
polymerization (ROP), the use of α,ω-epoxy esters allows the
introduction of a variety of groups through simple dicarbodii-
mide coupling that are now linked to the parent polymer
backbone via degradable ester linkages; (2) ring opening of the
epoxide results in a substituted poly(ethylene glycol) (PEG)-
like backbone that can be deemed biocompatible.30 In this
study, the introduction of alkyne ester groups along the pCL
and poly(dl-lactide) (pLA) backbone using the alkyne epoxy
ester propargyl 3-methylpentenoate oxide (PMPO) is
presented (Scheme 1), and the postfunctionalization of these
polymers with a variety of organic dyes that have utility in
functional imaging is demonstrated. As a step toward their
application in targeted therapeutics, the polymers were
formulated into nanoparticles, and their cellular compatibility
was evaluated.
1
Figure 1. Comparisons of (A) H NMR and (B) IR spectra of the
vinyl premonomer (black) and the functionalized epoxide (red).
RESULTS AND DISCUSSION
■
Copolymerization of PMPO with εCL and LA. To
establish the generality of alkyne epoxy esters for modification
of the aliphatic polyester backbone, copolymerization of PMPO
with εCL and 3,6-dimethyl-1,4-dioxane-2,5-dione (LA) was
investigated. The chemical structures of the expected
copolymers are shown in Scheme 1. The polymerization
conditions were chosen to yield polymers with number-average
molecular weights (Mn) in the range of 8000 to 10 000 g/mol.
This molecular weight range was chosen because it is suitable
for the production of nanoparticles via the nanoprecipitation
Synthesis of α,ω-Epoxy Ester Monomer. The premo-
nomer propargyl 3-methylpentenoate (PMP) was synthesized
in quantitative yield by esterification of propargyl alcohol (PA)
with 2-methyl-4-pentenoic acid using N,N′-dicyclohexylcarbo-
diimide and 4-(dimethylamino)pyridine. Trace amounts of
dicyclohexylurea were removed during the final workup using
flash chromatography. The purity of the premonomer was
1
confirmed by H NMR analysis (Figure 1A, spectrum 3) and
elemental analysis. The epoxidation was carried out using a
molar excess of Oxone under buffered conditions at near-
neutral pH to preserve the ester bond in PMP. The overall
conversion efficiency was around 62%. However, recovery of
the unreacted PMP educt during purification and further
epoxidation resulted in quantitative conversion. 1H NMR
analysis (Figure 1A, spectrum 4) revealed that the alkyne group
remained unreacted, indicating selective epoxidation of the
terminal vinyl group. This was further verified by IR analysis
(Figure 1B), where a clear signal at 3300 cm−1 for the alkyne
C−H vibration of PMPO is observed, which proves the
presence of an alkyne functional group in the epoxide.
method31,32 for targeted therapeutics and imaging. Typical H
1
NMR spectra of the copolymers of PMPO with εCL and LA
are shown in Figure 2. NMR analysis showed the presence of
intact alkyne protons in both copolymers (Figure 2, peak
assignment g, δ = 5.2 and 5.4 ppm, respectively).
Optimization of the Copolymerization Conditions.
Typical copolymerizations of the α,ω-epoxy esters with cyclic
lactones have been carried out at 130 °C using Sn(Oct)2 and
benzyl alcohol (BnOH) as the initiator system.29 However, the
presence of the alkyne bond in the comonomer warranted a
study to establish the most optimal temperature for the
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1
Figure 2. H NMR spectra of copolymers of PMPO with εCL (blue)
and LA (red). The PMPO content in the copolymers was 8%.
copolymerization. The findings of this study are summarized in
Table 1. It is clear that a lower polymerization temperature of
a
Table 1. Optimization of the Copolymerization Conditions
b
Mn (SEC)
Mn
copolymer
T [°C]
[103 g/mol]
PDI [103 g/mol]
poly(PMPO-co-LA)
90
130
90
5.1
3.3
7.8
5.2
1.6
1.7
1.4
1.6
8.2
8.2
poly(PMPO-co-εCL)
12.0
12.0
130
a
The εCL/PMPO or LA/PMPO ratio was set to a theoretical value of
b
90:10, and the polymerization was carried out for 24 h. Theoretical
molecular weight calculated on the basis of an initiator/monomer ratio
of 1/100.
Figure 3. (A) PMPO incorporation efficiency and (B) dependence of
the copolymer mass on PMPO for two different copolymers. In (B),
the numbers in parentheses are the mol % incorporation of the
epoxide in the copolymer.
90 °C was still capable of affording polymers with the desired
molecular weight without an appreciable increase in the
polydispersity index (PDI). The PDI values of 1.4−1.7 were
in the range of what is expected from ROP and are indicative of
a relatively narrow molecular weight distribution.33 Interest-
ingly, the copolymers with εCL had higher Mn than the
copolymers with LA. This may be attributed to a difference in
kinetics due to the higher ring strain in the εCL monomer.34
PMPO Incorporation Efficiency and Its Effect on
Copolymer Molecular Weight. The linear density of
biologically relevant information is important for many
applications such as cellular targeting and imaging, as this
affects the efficiency with which a system can interrogate a cell
in the former case and the signal-to-noise ratio in the latter.
Although Emrick and co-workers26,27 reported greater than 25
mol % incorporation of α-propargyl-δ-valerolactone into a pCL
backbone, in general one limiting factor in the modification of
aliphatic polyester backbones through copolymerization with
derivatized lactones such as depsipeptides has been the poor
incorporation35 of the functional monomer. Therefore, a study
to understand the incorporation of PMPO in both εCL and LA
copolymers was undertaken using feed ratios between 5 and 30
mol %. Furthermore, these feed ratios were chosen because past
studies have shown that beyond 30 mol % the polymer
molecular weights are severely diminished. The results for the
incorporation of PMPO into the polymer backbone are shown
in Figure 3.
The PMPO incorporation was estimated using the 1H NMR
integration by comparing the integrals of the alkyne protons
with those of methylene protons from the εCL backbone
(Figure 2 top, peak assignment 5, δ = 4.04 ppm) or the
methine protons of the LA unit (Figure 2 bottom, peak
assignment 1, δ = 5.17 ppm). As expected, increasing the
epoxide monomer in the feed leads to increased incorporation,
with a maximum incorporation of around 10%. The lack of a
one-to-one correlation between the feed ratio and the
composition in the copolymer can be attributed to the poor
self-reactivity of the α,ω-epoxy ester monomer as reported
earlier.29 However, with higher incorporation, a decrease in
molecular weight and yield is observed, and this is consistent
with past reports29 (Table S1 in the Supporting Information).
The incorporation in the α,ω-epoxy ester system is less than
what was reported by Emrick and co-workers, where the
similarity in cyclic ring structure between εCL and
valerolactone may have favored cross-reactivity between the
monomers, which is necessary for high incorporation. However,
in the system reported by Emrick, high incorporation comes at
the expense of crystallinity, as these copolymers are viscous
solids at room temperature. In contrast while α,ω-epoxy esters
have poor self-reactivity, they have good cross-reactivity with
cyclic lactones and result in copolymers with a well-defined
statistical distribution of epoxy ester units;29 more importantly,
they retain the thermal characteristics of the pCL or pLA
backbone, as discussed later. Thus, this system nevertheless
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yields copolymers of degradable aliphatic polyesters with a high
incorporation of clickable acetylene groups with narrow PDI
and acceptable molecular weights that are in the range suitable
for nanoparticle synthesis.32
Thermal Characteristics of Copolymers. Since the
intended application of these polymers is in injectable
nanoparticle systems, stability of the polymers both during
storage and use is a prerequisite. Therefore, the thermal
behavior of the copolymers was investigated to gain insights
into the changes in physical properties in comparison with pure
polyester. The decomposition temperatures (Td) of the
copolymers were in the ranges of 333−360 °C for poly-
(PMPO-co-εCL) and 238−251 °C for poly(PMPO-co-LA),
which are comparable to the Td values of the homopolymers
(pCL, 346 °C; pLA, 252 °C), suggesting that the copolymers
do not have any deficit in thermal stability in comparison with
the homopolymers. Differential scanning calorimetry analysis
revealed a decrease in the glass transition temperature (Tg) for
the copolymers in comparison with the homopolymers (Table
2). The homopolymers of εCL and LA synthesized under the
Table 2. Thermal Properties of Alkyne-Functionalized
Copolymers
PMPO in copolymer
[mol %]
Tg
[°C]
Tm
[°C]
Td
[°C]
copolymer
poly(PMPO-co-εCL)
0
2
−61
−59
−57
−51
42
59
50
346
359
360
333
252
251
248
238
4
50
8
40
poly(PMPO-co-LA)
0
147
143
142
138
2
41
4
39
10
28
polymerization conditions possessed Tg values of −58 and 42
°C, respectively, which are consistent with literature
reports.36,37 However, in the copolymers with greater than
8% incorporation of PMPO, a significant shift in Tg to lower
values [−67 and 28 °C for poly(PMPO-co-εCL) and
poly(PMPO-co-LA), respectively] was observed. This is an
expected outcome, as both backbone substitution and lower
molecular weight would promote polymer chain flexibility and
backbone rotation, both of which contribute to lowering of
Tg.38 However, at 10 mol % incorporation in the pLA system,
the Tg is significantly below physiological temperature. This
may prove valuable because functional polymers that undergo
softening when introduced into a physiological environment
could be used to leverage NP shape in delivery into cells.39
Modification of Copolymers by Click Reaction: Proof-
of-Principle Study Using Organic Dyes. Having demon-
strated that clickable groups can be incorporated into the
polymer backbone, it is important that these groups are
accessible for derivatization. Toward this objective, a study was
undertaken to investigate the click reactions between poly-
(PMPO-co-LA) and several organic dyes (Figure 4A).
Figure 4. (A) Schematic of copolymers clicked with different organic
dyes. (B) Comparison of the 1H NMR spectra of poly(PMPO-co-LA)
before (red) and after (black) the click reaction with 7-HC
(conversion ∼57%). (C) Summary of the conversion efficiencies of
the click reactions with various dyes. The concentrations of the dyes
along the polymer backbone were determined from calibration curves
over a concentration range of 2−50 μg (Figure S4 in the Supporting
Information). (D) Example UV−vis spectra of dye-modified
copolymer systems. Values shown next to the peaks are λmax values
for the dyes clicked to the copolymer; the λmax values for the free dyes
are 552 nm for Cy3-Azide and 740 nm for Atto740. (E) Plot showing
linear scaling of the fluorescence intensity of the dye-modified
copolymers.
As a first example, the reaction of the copolymer (3.8 mol %
alkyne groups) with 3-azido-7-hydroxycoumarin (7-HC), a dye
that becomes fluorescent upon conversion to the triazole
derivative, was investigated. In this system, the appearance of
fluorescence provides direct confirmation of the formation of
the 1,3 bipolar adduct and hence quantification of the click
reaction. The 1H NMR spectrum of the clicked 7-HC
copolymer conjugate (Figure 4B) clearly showed signals
corresponding to the newly formed 1,2,3-triazole ring (peak
assignments a−e), and additionally, this click modification
showed >50% efficiency (i.e., conversion of the alkyne groups),
indicating good accessibility of the alkyne moieties to chemical
modification in solution.
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To elaborate this to other organic dyes that have utility in cell
and functional imaging, click reactions of poly(PMPO-co-εCL)
(2 mol % alkyne groups) with azide-functionalized cyanine 3
(Cy3) and ATTO-740 were studied. These dyes were chosen
because they have different adsorption (λabs,Cy3 = 552 nm,
λabs,ATTO‑740 = 740 nm) and emission (λem,Cy3 = 570 nm,
λem,ATTO‑740 = 764 nm) wavelengths. Furthermore, while Cy3 is
widely used in cell imaging, ATTO-740 is a near-infrared dye
that is suitable for functional fluorescence imaging. The
conversion efficiency (i.e, the ratio of the molar equivalents
of dye on the polymer backbone to the initial dye
concentration) is summarized in Figure 4C. Conversions as
high as 80% could be achieved with the Cy3-Azide system. The
ability to accomplish derivatization of the copolymer backbone
with a variety of organic dyes was verified by UV−vis
spectroscopy (Figure 4D). The excellent correspondence
between the absorption maxima of the Cy3- and ATTO-740-
conjugated polymers and those of the free dyes suggests the
absence of any dye quenching upon covalent immobilization,
which was also confirmed by the linear scaling of the adsorption
as a function of polymer concentration (Figure 4E). The
absence of any drastic change in molecular weight or an
increase in PDI upon copolymer functionalization (Table S2 in
the Supporting Information) is suggestive of minimal
degradation during modification.
concentration of 0.5 mg/mL, a high cell viability of 80% was
observed, suggesting that the dye-modified copolymers have
good cytocompatibility.
Cytocompatibility of Dye-Functionalized Nanopar-
ticles. In order to ascertain the suitability of these novel dye-
functionalized copolymers for injectable and imaging applica-
tions, dye-functionalized pCL copolymers were formulated into
NPs using the single-step nanoprecipitation method.32
Independent of the dye modification, all three copolymers
yielded stable NPs that exhibited size distributions that are
acceptable for injectable applications40 and comparable zeta
potentials (Table 3) and showed good stability even after 10
a
Table 3. Overview of Synthesized Nanoparticles
Figure 5. Cytocompatibility of dye-labeled NPs: (A) Schematic
showing NPs synthesized from single-dye-labeled copolymers and a
blend of three individually labeled copolymers. (B) Viability of HeLa
cells in the presence of dye-labeled NPs as determined using MTT
assay after 24 h of incubation. The results are reported as values
normalized to cell viability on tissue-culture plastic. (C) Fluorescence
microscopy images of HeLa cells incubated with NPs produced by
blending of three copolymers individually labeled with 7-HC, Cy3-
Azide, and ATTO-740. For comparison, images of HeLa cells
incubated with single-dye (Cy3 or 7-HC)-functionalized NPs are
shown in Figure S5 in the Supporting Information.
size [nm]
zeta potential
[mV]
copolymer
poly(PMPO-co-εCL)
(n = 3)
140
2
−27
−7
3
1
poly(PMPO-co-εCL) clicked with Cy3-
Azide
138 10
poly(PMPO-co-εCL) clicked with
ATTO-740
150 22
−24
7
poly(PMPO-co-εCL) clicked with 7-HC
130 10
160 15
−11
−28
3
5
poly(PMPO-co-εCL) with all three dyes
blended
a
Concentration of polymer 0.5% (w/v) (5 ng/mL).
The visualization of fluorescent signals emanating from all
three dyes in a single NP system was explored by incubating
HeLa cells with NPs prepared from a blend of three single-dye-
functionalized copolymers: 7-HC, Cy3-Azide and ATTO-740
(Figure 5C). It is evident that the NPs can be clearly visualized
within the cells in all three respective fluorescence channels,
indicating that the excitation and emission are not impacted by
the presence of multiple fluorophores with the NPs. This bodes
well for the exploration of these polymers for the development
of imaging and diagnostic probes.
days, with a maximum size change of 10% (Table S3 in the
Supporting Information). Furthermore, blending of the single-
dye-functionalized copolymers (Figure 5A) afforded NPs
possessing multiple chromophores without compromising the
size and surface charge characteristics. NPs bearing multiple
distinct spectral signatures can be useful in optically coded
systems for diagnostics or imaging based on optical multi-
plexing.
Utility of Dye-Functionalized NPs for Imaging in Vivo.
Toward the application of these copolymers in targetable
therapeutics and imaging, the cytotoxicity of the NPs in HeLa
cells was studied. The viabilities of HeLa cells incubated with 7-
HC-, Cy3-Azide-, and ATTO-740-modified NPs at different
concentrations of 0.025, 0.125, and 0.5 mg/mL after 24 h of
incubation are shown in Figure 5B. Even at the highest NP
In cellular and tissue imaging, the ability to accurately
superimpose the information from various modalities can
provide exceptional functional information.41 This is achieved
using nanoprobes bearing information that can be interrogated
by multiple modalities such as magnetic resonance imaging, X-
ray computer tomography, and fluorescence imaging.42
Polymers are particularly well suited for fashioning such
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nanoprobes as they can be tailored to carry a plurality of agents.
In the conventional approach, the fluorescent molecules are
encapsulated in a nanocarrier along with other agents. In this
scenario, leakage of the fluorescent molecule beyond the
confines of the nanocarrier can confound the acquisition and
assignment of information. In this context, advantages that
covalent immobilization of the dye can confer over the
conventional approach of encapsulation are higher signal-to-
noise ratio and improved localization of the fluorescence. To
explore this premise, coumarin 343 (C-343), a dye with very
high affinity for cell lipids, was incorporated into NPs through
encapsulation and click-chemistry modification, and the
distributions of fluorescence in HeLa cells were examined
after 2 h. First, we observed that in comparison with NPs
prepared from polymers with covalently immobilized C-343,
encapsulation of C-343 resulted in a 60% increase in NP size
(Figure 6A), which is not desirable because size can profoundly
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental methods and characterization, synthetic details
for preparation of 7-HC, overview of alkyne-functionalized
copolymers, calibration curves for Cy3-Azide and ATTO-740
to calibrate the concentrations of the dyes along the polymer
backbone, molecular weights and PDI values for unmodified
and dye-functionalized copolymers, stability studies of NPs
sizes (comparison of day 1 to day 10), and fluorescence
microscopy images of HeLa cells coincubated with single-dye-
functionalized NPs. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
prasad.shastri@gmail.com
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Carina Knittel and Esther Kohler for
assistance and Dr. Ulrike Shastri for careful reading of the
manuscript. This work was funded by the Fifth INTERREG
Upper Rhine Program (Project A21: NANO@MATRIX), the
Excellence Initiative of the German Federal and State
Governments (Grant EXC 294, Centre for Biological Signalling
Studies), and the University of Freiburg.
REFERENCES
■
(1) Shoichet, M. S. Macromolecules 2010, 43, 581−591.
(2) Kroeze, R. J.; Helder, M. N.; Govaert, L. E.; Smit, T. H. Materials
(Basel) 2009, 2, 833−856.
(3) Shin, H.; Jo, S.; Mikos, A. G. Biomaterials 2003, 24, 4353−4364.
(4) Sinha, V. R.; Bansal, K.; Kaushik, R.; Kumria, R.; Trehan, A. Int. J.
Pharm. 2004, 278, 1−23.
(5) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347−360.
(6) Freed, L. E.; Vunjak-Novakovic, G.; Biron, R. J.; Eagles, D. B.;
Lesnoy, D. C.; Barlow, S. K.; Langer, R. Nat. Biotechnol. 1994, 12,
689−693.
(7) Nair, L. S.; Laurencin, C. T. Prog. Polym. Sci. 2007, 32, 762−798.
(8) Langer, R. Acc. Chem. Res. 2000, 33, 94−101.
(9) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M.
Chem. Rev. 1999, 99, 3181−3198.
(10) Meng, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2009, 10,
197−209.
(11) Mundargi, R. C.; Babu, V. R.; Rangaswamy, V.; Patel, P.;
Aminabhavi, T. M. J. Controlled Release 2008, 125, 193−209.
(12) Lee, S.; Jeong, J.; Shin, S.; Kim, J.; Chang, Y.; Chang, Y.; Kim, J.
J. Magn. Magn. Mater. 2004, 276, 2432−2433.
(13) Albertsson, A.-C.; Varma, I. K. Biomacromolecules 2003, 4,
1466−1486.
(14) Xu, L.; Zhang, Z.; Wang, F.; Xie, D.; Yang, S.; Wang, T.; Feng,
L.; Chu, C. J. Colloid Interface Sci. 2013, 393, 174−181.
(15) Cheng, Y.; Hao, J.; Lee, L. A.; Biewer, M. C.; Wang, Q.; Stefan,
M. C. Synthesis 2012, 44, 2163−2173.
Figure 6. C-343-dye-functionalized NPs: (A) Size and zeta potential of
clicked and blended C-343-functionalized NPs. (B) Fluorescence
microscopy images of HeLa cells coincubated with C-343-dye-
functionalized NPs: (I) dye covalently bonded; (II) dye blended
into the copolymer.
impact biodistribution.43 However, more significantly, fluo-
rescence microscopy revealed that while the fluorescence in
cells incubated with NPs with encapsulated dye was feeble and
dispersed throughout the cell, in contrast the fluorescence in
cells treated with NPs with covalently immobilized dye showed
highly punctate, intense fluorescence that was remarkably
confined to the cytosol, with very negligible staining of the cell-
membrane lipids (Figure 6B).
SUMMARY
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(16) Guo, J.; Gao, X.; Su, L.; Xia, H.; Gu, G.; Pang, Z.; Jiang, X.; Yao,
L.; Chen, J.; Chen, H. Biomaterials 2011, 32, 8010−8020.
(17) El Habnouni, S.; Darcos, V.; Coudane, J. Macromol. Rapid
Commun. 2009, 30, 165−169.
A new approach for the synthesis of aliphatic degradable
polyesters with a high density of alkyne functionality has been
presented. The accessibility of the pendant alkyne functional
groups to solution-phase click chemistry has been demon-
strated using various fluorescent organic dyes that have broad
utility in cell and tissue imaging. This class of dye-function-
alized polyesters possesses good cytocompatibility and offers
potential advantages over conventional systems for the
development of nanoprobes for cellular and tissue imaging.
(18) Nottelet, B.; El Ghzaoui, A.; Coudane, J.; Vert, M.
Biomacromolecules 2007, 8, 2594−2601.
(19) Ponsart, S.; Coudane, J.; Vert, M. Biomacromolecules 2000, 1,
275−281.
(20) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004−2021.
F
dx.doi.org/10.1021/ja505629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(21) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.;
Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 16793−16797.
(22) Lu, C.; Shi, Q.; Chen, X.; Lu, T. J. Polym. Sci., Part A: Polym.
Chem. 2007, 45, 3204−3217.
́ ̂ ́ ̂
(23) Riva, R.; Schmeits, S.; Stoffelbach, F.; Jerome, C.; Jerome, R.;
Lecomte, P. Chem. Commun. 2005, 5334−5336.
(24) Silvers, A. L.; Chang, C.-C.; Emrick, T. J. Polym. Sci., Part A:
Polym. Chem. 2012, 50, 3517−3529.
(25) Maji, S.; Zheng, M.; Agarwal, S. Macromol. Chem. Phys. 2011,
212, 2573−2582.
(26) Parrish, B.; Breitenkamp, R. B.; Emrick, T. J. Am. Chem. Soc.
2005, 127, 7404−7410.
(27) Cooper, B. M.; Emrick, T. J. Polym. Sci., Part A: Polym. Chem.
2009, 47, 7054−7065.
(28) Shastri, V. P. U.S. Patent 6,730,722 B2, 2004.
(29) Wurth, J. J.; Shastri, V. P. J. Polym. Sci., Part A: Polym. Chem.
2013, 51, 3375−3382.
(30) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Angew.
Chem., Int. Ed. 2010, 49, 6288−6308.
(31) Schneider, J.; Jallouk, A. P.; Vasquez, D.; Thomann, R.; Forget,
A.; Pino, C. J.; Shastri, V. P. Langmuir 2013, 29, 4092−4095.
(32) Sussman, E. M.; Clarke, M. B.; Shastri, V. P. Langmuir 2007, 23,
12275−12279.
(33) Dechy-Cabaret, O.; Martin-Vaca, B.; Bourissou, D. Chem. Rev.
2004, 104, 6147−6176.
(34) Saiyasombat, W.; Molloy, R.; Nicholson, T. M.; Johnson, A. F.;
Ward, I. M.; Poshyachinda, S. Polymer 1998, 39, 5581−5585.
(35) Barrera, D. A.; Zylstra, E.; Lansbury, P. T.; Langer, R. J. Am.
Chem. Soc. 1993, 115, 11010−11011.
(36) Labet, M.; Thielemans, W. Chem. Soc. Rev. 2009, 38, 3484−
3504.
(37) Garlotta, D. J. Polym. Environ. 2002, 9, 63−84.
(38) Tieke, B. Makromolekulare Chemie; Wiley-VCH: Weinheim,
Germany, 2005.
(39) Yoo, J.; Mitragotri, S. Proc. Natl. Acad. Sci. U.S.A. 2010, 107,
11205−11210.
(40) Voigt, J.; Christensen, J.; Shastri, V. P. Proc. Natl. Acad. Sci.
U.S.A. 2014, 111, 2942−2947.
(41) Vonwil, D.; Christensen, J.; Fischer, S.; Ronneberger, O.;
Shastri, V. P. Mol. Imaging Biol. 2013, 16, 350−361.
(42) Baker, M. Nature 2010, 463, 977−980.
(43) Gaumet, M.; Vargas, A.; Gurny, R.; Delie, F. Eur. J. Pharm.
Biopharm. 2008, 69, 1−9.
G
dx.doi.org/10.1021/ja505629w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX