A facile method to clickable sensing polymeric nanoparticles

Article abstract of DOI:10.1039/b914358k

Clickable biocompatible nanoparticles were prepared in a one-pot process by microemulsion polymerization using acrylamide, N,N′-methylene bisacrylamide and either N-(11-azido-3,6,9-trioxaundecanyl)acrylamide or N-propargylacrylamide, which were then readi

Full text of DOI:10.1039/b914358k

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A facile method to clickable sensing polymeric nanoparticlesw
Katharina Welser,ab M. D. Ayal Perera,a Jonathan W. Aylott*b and Weng C. Chan*a
Received (in Cambridge, UK) 17th July 2009, Accepted 28th August 2009
First published as an Advance Article on the web 15th September 2009
DOI: 10.1039/b914358k
Clickable biocompatible nanoparticles were prepared in a
one-pot process by microemulsion polymerization using acrylamide,
N,N0-methylene bisacrylamide and either N-(11-azido-3,6,9-
trioxaundecanyl)acrylamide or N-propargylacrylamide, which
were then readily modified by CuAAC reaction to afford sensing
nanomaterials.
N,N0-methylene bisacrylamide
methacrylamide 3 (see Scheme 1).5,6
2 and N-(3-aminopropyl)-
We observed that these amine-bearing nanoparticles
aggregate over time, resulting in limited shelf-lives (less than
one month) even when stored under argon at ꢀ18 1C. This
aggregation, confirmed by dynamic light scattering (DLS), is
postulated to be the result of a slow Michael addition reaction
between the free amine group and unreacted 2 on the surface
of adjacent particles.7 We rationalized that we could increase
the stability and thus prolong the shelf-life of the nanoparticles
by replacing the amine functionality by clickable azide
(essentially a masked amine) and alkyne handles, which will
be achieved by the use of appropriate monomer units. Thus,
the required monomers 4 and 5 were readily obtained in
moderate yields by the reaction of acryloyl chloride with
11-azido-3,6,9-trioxaundecan-1-amine and propargylamine,
respectively according to a modified literature procedure8
(see ESIw). The monomers 4 and 5 were directly incorporated
in the nanoparticles during the polymerization reaction
(Scheme 1), thus making a secondary functionalization step
after assembly of the nanoparticles unnecessary. Different
formulations were tested and it was found that by using a
weight percentage of less than 3.5% of either 4 or 5 in the
monomer mixtures, unimodal particles were obtained which
were on average 25 nm in diameter (Fig. 1a). Interestingly,
bimodal distributions or significantly larger particles
(ca. 400 nm in diameter) were obtained when higher amounts
of 4 and 5 were used in the polymerization reactions.
‘Smart functional nanomaterials’ have attracted considerable
interest over the past decade, due to their unique behavior that
can be exploited in a wide range of applications such as
diagnostics, targeted drug delivery and nanomedicine. Access
to these smart nanomaterials can be facilitated by the Huisgen
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction,
which is commonly regarded as the prime example of click
chemistry since it is chemoselective and can be performed in
aqueous medium at a moderate temperature.1 Notably,
CuAAC has sparked a re-invigoration of interest in the
development of nanoparticulate bioconjugation tools. Both
azide and alkyne groups are stable in aqueous solutions and
can be used as inert handles which further serve as multi-
functional platforms for targeted delivery, biodetection and
diagnosis.2 Recently, mini-emulsion and emulsion polymerizations
have been reported as useful en route to prepare colloidally
stable aqueous dispersions of clickable nanoparticles.3
However, these polymerizations are typically carried out at
relatively high temperatures (70–90 1C), which makes it
difficult to introduce the temperature sensitive azide moiety
in a one pot procedure. Consequently, the alkyne handle is
installed following polymerization and nanoparticle formation.3
Here, we report a versatile one-pot procedure for the
synthesis of both azido- and alkynyl-functionalized polymeric
nanoparticles at room temperature. To illustrate their utility
as sensing nanomaterials, CuAAC reactions were used to
transform these click-readied nanoscale substrates into
pH- and protease responsive systems.
The successful introduction of the azido groups was demon-
strated by the appearance of a characteristic absorbance at
2110 cmꢀ1 in the FTIR-spectrum (Fig. 1b). However, the
characteristic absorption of alkynyl groups (expected at
around 3300 cmꢀ1) could not be detected by FT-IR due to
strong absorbance of the polymer backbone; the alkyne
moiety was nevertheless observed by Raman spectroscopy
(see Fig. S1, ESIw). As anticipated, the clickable nanoparticles
showed significantly longer shelf lives (over 2 months) when
stored at ꢀ18 1C, compared to the amine-functionalized ones
(see Fig. S2, ESIw).
The availability and reactivity of the functional groups in
these nanoparticles toward CuAAC were then established by
reaction with complementary clickable entities. Specifically,
the nanoparticles carrying click reactive functionalities were
allowed to react with either a commercially available alkynyl-
functionalized TAMRA dye 6 or an azide-functionalized
fluorescent peptide 7 in the presence of the Cu(I) catalyst
(7–10 mol%) and the ligand TBTA9 (7–10 mol%). The
reactions were allowed to proceed for 48 h at ambient
temperature, after which time the nanoparticles were purified
by successive washing with DMF and EtOH.
Our synthetic strategy to these functionalized nanoparticles
is based on a room temperature microemulsion polymerization
technique, which is known to produce nm-sized particles
with a narrow size distribution.4 Our earlier work to add
chemical functionality to polymeric nanosensors was focused
on the use of amine-functionalized polyacrylamide nano-
particles which are derived from acrylamide 1, the crosslinker
a School of Pharmacy, Centre for Biomolecular Sciences,
University of Nottingham, University Park, Nottingham,
UK NG7 2RD. E-mail: weng.chan@nottingham.ac.uk
b School of Pharmacy, Boots Science Building,
University of Nottingham, University Park, Nottingham,
UK NG7 2RD. E-mail: jon.aylott@nottingham.ac.uk
w Electronic supplementary information (ESI) available: Experimental
protocols and products characterization. See DOI: 10.1039/b914358k
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Scheme 1 Synthetic strategies for the preparation of different functionalized NPs via inverse microemulsion polymerization. The CuAAC
chemistry is then used to attach functional molecules to the particles.
leaching of the fluorophore from the nanoparticle. These
hybrid nanoparticles were then clicked to reagent 7, which
contained the pH-sensitive fluorophore 5-carboxyfluorescein
(5-FAM). The resulting nanosensors were successfully
calibrated against a glass electrode over a pH range of 5.3 to
7.6 in phosphate buffer saline (PBS), giving a calibration curve
analogous to that defined in the literature for pH nanosensors
utilizing fluorescein and rhodamine derivatives (Fig. 3).10
Next, the protease responsive nanosensor was afforded
Fig. 1 (a) DLS of azide functionalized NPs (black square) and alkyne
by clicking the fluorogenic substrate Z-Gly-Gly-Leu-ACA-
Lys(Ne-4-pentynoyl)-NH2
8 to the azido-bearing nano-
functionalized NPs (blue triangle). (b) FTIR spectra of blank
(prepared using the monomers 1 and 2; blue solid line) and azide
particles (Scheme 2). The chosen peptide contains a sequence
domain that is recognized by subtilisin, an enzyme widely used
for studying enzyme–substrate interactions and was thus
employed as the model protease.11 We selected the hetero-
bifunctional 7-aminocoumarin-4-acetic acid (ACA) 9 as the
fluorophore. The fluorescence of 9 was previously established
to be considerably quenched when the anilino group is
acylated as is the case when it is conjugated to the C-terminus
of a peptide domain.6 The fluorescence is recovered upon
proteolytic cleavage caused by the liberation of the anilino
group. Furthermore, the reagent 9 is synthetically accessible in
bulk from a key intermediate to our previously reported green
fluorescent coumarin dyes.6
functionalized NPs (black solid line).
The success of the CuAAC reaction was confirmed by
the detection of a noticeable fluorescence signal when the
nanoparticle suspensions comprising conjugated 6 and 7 were
excited at lex = 555 nm and lex = 490 nm, respectively
(Fig. 2). Importantly, the controls exhibited no significant
fluorescence, thus indicating that the fluorescent counterparts
were actually ‘clicked’ onto the nanoparticles and not just
physically adsorbed. Gratifyingly, no noticeable differences in
the nanoparticle size were observed, which indicates that the
nanoparticle dimensions were preserved following the CuAAC
(see Fig. S3, ESIw).
Because of the poor nucleophilicity of the anilino group
of 9, it was possible to regioselectively react the fluorophore
under mild coupling conditions to a Lys(ivDde)-preloaded
Rink-amide Novagelt polymer support. The assembly of the
peptide using a standard Fmoc/t-Bu solid-phase peptide
synthesis method then proceeded smoothly.12 Following
removal of the ivDde protecting group with 5% hydrazine
in DMF,13 the alkyne functionality was installed by
reaction with HATU/HOAt/DIPEA-activated 4-pentynoic
In order to further illustrate the conjugation capacity of our
new clickable nanoparticles, we bestowed the nanoparticles
with unique pH and protease responsive features. With the
aim to design ratiometric pH-responsive nanosensors, the
alkyne-bearing nanoparticles were prepared in an inverse
microemulsion, simultaneously incorporating the reference
dye TAMRA which is conjugated to dextran (see ESIw).
Dextran-bound TAMRA (Mw = 10 000) was used to prevent
Fig. 3 (a) Emission spectra of pH-responsive nanosensors recorded
at different pH values (lex = 490 nm and 555 nm for 5-FAM
Fig. 2 Emission spectra of nanoparticulate products showing successful
CuAAC-mediated conjugation of (a) 6 to azide functionalized NPs
and (b) 7 to alkyne modified NPs.
and TAMRA, respectively). (b) Plot of intensity ratios (lem,5-FAM
520 nm/lem,TAMRA = 580 nm) vs. pH values.
=
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Fig.
4
(a) Subtilisin mediated cleavage reaction of 8-modified
NPs (lex = 380 nm) and (b) enzyme kinetics at different subtilisin
concentrations.
Scheme 2 The synthesis of subtilisin responsive nanosensors.
which complementary clickable moieties can be coupled by
CuAAC reaction, should find applications in the design of
biodetection and diagnostic systems.
acid. Subsequently, acidolytic treatment of the resin-bound
peptidic construct gave the required native peptide substrate in
good yield and purity (see ESIw).
KW would like to acknowledge Engineering & Physical
Sciences Research Council and the Royal Society of
Chemistry, UK for the funding of an analytical science
studentship.
The subtilisin responsive nanosensor was then obtained by
CuAAC-mediated conjugation of the bioorthogonal alkynyl-
bearing peptide substrate 8 with the complementary azido-
bearing nanoparticle (see ESIw). As anticipated, a significant
and rapid increase in fluorescence was observed when the
constructed nanosensor was incubated with different
concentrations of subtilisin at 37 1C in Tris-HCl buffer
(pH = 8.20) (Fig. 4a). In fact, after 1 min, about an 80-fold
rise in the fluorescence was observed when subtilisin was
present at a concentration of 10 mM, indicating a fast initial
protease response (Fig. 4b). Control experiments revealed that
no significant change in the fluorescence was observed in
the absence of subtilisin or in the presence of chymotrypysin.
It is evident that the subtilisin-responsive nanosensor
presented here could easily be re-engineered for the detection
of other endopeptidases by choosing the appropriate clickable
substrates. Moreover, it is noteworthy that in this new
conjugation strategy, there is no need for the protection of
reactive functional groups in the substrate domain due to the
chemoselectivity of the CuAAC reaction.
Notes and references
1 (a) H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem.,
Int. Ed., 2001, 40, 2004; (b) C. W. Tornøe, C. Christensen and
M. Meldal, J. Org. Chem., 2002, 67, 3057; (c) W. H. Binder and
R. Sachsenhofer, Macromol. Rapid Commun., 2008, 29, 952;
(d) M. Meldal and C. W. Tornoe, Chem. Rev., 2008, 108, 2952.
2 C. D. Hein, X. M. Liu and D. Wang, Pharm. Res., 2008, 25, 2216.
3 C. E. Evans and P. A. Lovell, Chem. Commun., 2009, 2305.
4 Surfactants and Polymers in Aqueous Solution, ed. K. Holmberg,
B. Jonsson, B. Kronberg and B. Lindman, John Wiley & Sons,
Chichester, 2003.
5 (a) A. Webster, S. J. Compton and J. W. Aylott, Analyst, 2005,
130, 163; (b) P. G. Coupland, S. J. Briddon and J. W. Aylott,
Integr. Biol., 2009, 1, 318.
6 K. Welser, J. Grilj, E. Vauthey, J. W. Aylott and W. C. Chan,
Chem. Commun., 2009, 671.
7 B. M. Moore, PhD Thesis, University of Nottingham, 2008.
8 M. Malkoch, R. J. Thibault, E. Drockenmuller, M. Messerschmidt,
B. Volt, T. P. Russell and C. J. Hawker, J. Am. Chem. Soc., 2005,
127, 14942.
9 T. R. Chan, R. Hilgraf, K. B. Sharpless and V. V. Fokin,
Org. Lett., 2004, 6, 2853.
10 H. Sun, A. M. Scharff-Poulsen, H. Gu and K. Almdal,
Chem. Mater., 2006, 18, 3381.
11 Y. Kanaoka, T. Takahashi, H. Nakayama and K. Tanizawa,
Chem. Pharm. Bull., 1985, 33, 1721.
The approaches outlined herein clearly demonstrate the
opportunities offered by our new clickable nanoparticles to
create unique and flexible sensing architectures, in which
additional functionalities or sensing modules could be readily
installed using the robust CuAAC reaction.
In conclusion, we have established
a facile one-pot
12 Fmoc Solid Phase Synthesis: A Practical Approach, ed. W. C. Chan
and P. D. White, Oxford University Press, Oxford, 2000.
13 S. R. Chhabra, B. Hothi, D. J. Evans, P. D. White, B. W. Bycroft
and W. C. Chan, Tetrahedron Lett., 1998, 39, 1603.
procedure for the synthesis of water dispersible clickable
polymeric nanoparticles using a microemulsion polymerization
technique. These customized and tunable nanoparticles, to
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Chem. Commun., 2009, 6601–6603 | 6603