Strain release in organic photonic nanoparticles for protease sensing

Article abstract of DOI:10.1021/ja301259v

Proteases are overexpressed in most cancers and proteolytic activity has been shown to be a viable marker for cancer imaging in vivo. Herein, we describe the synthesis of luminescence-quenched shell cross-linked nanoparticles as photonic nanoprobes for protease sensing. Protease sensing scheme is based on a "turn-on" mechanism where the protease cleaves peptide cross-linkers of the fluorescence-quenched shell cross-linked NP (OFF state) leading to a highly emissive non-cross linked NP (ON state). The cross-linked particles can be strained by exposure to a good solvent and proteolysis allows for particle expansion (swelling) and a recovery of the luminescence.

Full text of DOI:10.1021/ja301259v

Communication
pubs.acs.org/JACS
Strain Release in Organic Photonic Nanoparticles for Protease
Sensing
Carlos Cordovilla and Timothy M. Swager*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: Proteases are overexpressed in most cancers
and proteolytic activity has been shown to be a viable
marker for cancer imaging in vivo. Herein, we describe the
synthesis of luminescence-quenched shell cross-linked
nanoparticles as photonic nanoprobes for protease sensing.
Protease sensing scheme is based on a “turn-on”
mechanism where the protease cleaves peptide cross-
linkers of the fluorescence-quenched shell cross-linked NP
(OFF state) leading to a highly emissive non-cross linked
NP (ON state). The cross-linked particles can be strained
by exposure to a good solvent and proteolysis allows for
particle expansion (swelling) and a recovery of the
luminescence.
Figure 1. (a) Schematic representation of NPs structure. (b) The ON-
and OFF-states and relationship of cross-linking in the other polymer
shell of the NPs. (c) Protease triggered “turn on” sensing scheme.
he diagnosis of cancer at its earliest stages is critical to
obtain optimal treatment. New imaging methods that
detect smaller malignant cancer tumors have the potential to
dramatically improve our ability to fight this disease. Optical
imaging methods with inorganic nanoparticles,1 organic dye-
containing nanoparticles,2 single wall nanotubes (SWNT),3 and
metal nanoclusters4 have been developed.
T
conjugated polymers is the exceptionally large optical
absorbance of these structures, coupled with their ability to
behave as antennas, effectively collecting the excitations and
providing for high signals.11,12 This sensitivity can ultimately
enable imaging technologies using moderate laser powers.
As we detail here, our NP design involves the assembly of an
“ABCBA” pentablock polymer structure wherein the central
“C” block is a semiconducting PPE. The terminal “A” hydrogel
block provides the hydrophilicity needed for in vivo studies and
prevents aggregation of the NPs under physiological conditions.
The “B” block is designed to be reacted post-NP formation to
incorporate functional groups that produce transduction events
that affect the particle’s emission properties.
Aggregation induced interactions of π-systems of semi-
conducting polymers often lead to fluorescence self-quenching
processes as a result of energy transfer to weakly emitting
excimer-type associations between individual chains.10a Accord-
ingly, a subtle tuning of the π-associations in the core of the
nanoparticle controls the emission intensity of the NPs. Taking
advantage of this property, we have developed a luminescence
“turn ON”−“turn OFF” system. Our designs involve the
formation of NPs with tightly packed PPEs that are trapped in
partially fluorescence quenched state by cross-linking the other
shells of NPs (OFF state) by reaction of external succinimide
groups in the B-polymer block with a short peptide (Figure
1b). For the protease sensing reported herein, we have used the
following sequence [H]-Lys-Cys-Arg-Pro-Leu-Ala-Leu-Trp-
Arg-Ser-Lys-[NH2] as a shell cross-linking agent. A protease
Proteases are involved in cancer,5 AIDS, neurodegenerative
diseases, and inflammation. Proteolytic activity has been
recently proven as an effective marker for cancer imaging in
vivo.6 As a result, there is an increasing interest in protease
sensing as an alternative to other imaging techniques for cancer
detection. There are several examples of nanoprobes for in vivo
fluorescence7 and magnetic resonance8 monitoring of protease
activity. These sensing schemes are based upon the release of a
quencher/relaxation agents from the nanoprobe.
We report, herein, fluorescent organic nanoparticles (NPs) as
imaging probes for protease activity and demonstrate a new
strain-release transduction mechanism. Our NP design (Figure
1a) consists of a highly fluorescent core and an external
hydrogel coating displaying reactive functional groups. The
core is based on poly(phenylene ethynylene) (PPE) bearing
pentiptycene units that minimize interpolymer π-aggregation
and a minority (5% or less) far red emitting segment
(chromophore) inserted randomly into the PPE backbone.
Our choice of pentiptycene is based on their previously
demonstrated outstanding performance as sensors for the
detection of chemical and biological analytes,9 and explosives.10
A critical component of our design is that changes in the local
environment of the NPs will be amplified by the mobility of the
excitons, which enhances energy transfer (ET) to the
chromophores. An advantage of NPs based on organic
Received: February 8, 2012
Published: April 10, 2012
© 2012 American Chemical Society
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Communication
sensing scheme making use of our NPs is illustrated in Figure
1c and is a “turn-ON” mechanism wherein a NP is captured in a
nonequilibrium contracted (strained) OFF state, and when the
protease cleaves specific positions of the peptide sequence,13
the particle can expand to an emissive ON state.
sonication.15 The size of the nanoparticles was measured by
Dynamic Light Scattering (DLS) techniques. Depending upon
the initial concentration in the THF solution, it is possible to
tune the size of the nanoparticles over a wide range of
diameters. For biological applications, we have implemented
conditions to obtain dispersions of nanoparticles around 80 nm
in diameter with narrow size distributions.
The design of the semiconductive polymer NP core is critical
and the aggregation induced quenching is required to be readily
modulated. To balance the interpolymer forces and also make
use of energy transfer, we have made use of a pentiptycene
elaborated PPE developed in our group, which is copoly-
merized with a lower band-gap perylene minority emitter.
Although the pentiptycene PPEs are relatively immune from
aggregation induced quenching, the presence of the perylene
monomer results in segments that are capable of undergoing π-
associations leading to reduced (quenched) emission in
aggregated states. An appropriate perylene dye that functions
as a well-behaved monomer for the Sonogashira−Hagihara
polymerizations was used to create PPE. To this end, we
developed a functionalized perylene diacetylene 2 (see
Supporting Information) that displays a high photolumines-
cence quantum yield. A predictable amount of this fluorophore
can be readily inserted into the polymer backbone to serve as a
high efficiency emissive trap for excitons. The PPE-block was
synthesized by cross-coupling of the two different acetylenes,
namely, 2 and the pentiptycene diacetylene, and a slight excess
of a dialkoxy diiodoarene derivative. Quantitative incorpo-
rations of the perylene monomer (2) were achieved. This
allows a control of the amount of the low band gap perylene
emitter in the polymer between 0.5 and 5 mol % by easily
controlling the stoichiometry of the reaction. The well-behaved
nature of this polymerization is also confirmed by the fact that
these polymers display polydispersities similar to their non-
perylene containing analogues.10 End-capped materials (Figure
2) were obtained by quenching the polymerization reaction
The non-cross-linked NPs are highly emissive and their
emission spectra (Figure 3) show two peaks ascribed to the
Figure 3. (a) Photoluminescence spectra of aqueous non-cross linked
PPE-NHS nanoparticle suspensions containing different amounts of
dye (λex: 410 nm). (b) Aqueous non-cross linked PPE-NHS
nanoparticle suspensions under irradiation at 365 nm at different
dye loading (mol %).
conjugated polymer backbone (454 nm) and the emissive dye
(604 nm) maxima (Figure 3). NPs derived from copolymers
bearing perylene moieties show higher quantum yields than
those lacking this dye.16 We observe efficient energy transfer
(ET) from the PPE backbone to the perylene and the excitation
of the nanoparticle at the absorption maximum of the PPE
backbone (410 nm) results in a dominant perylene-based
emission at around 604 nm. The efficiency of this energy
transfer was quantified by measuring the ratio between the
fluorescence intensity emitted from the perylene traps (IAD
)
and the total emission intensity (I) of the nanoparticle.
Additionally, the enhancement in the 604 nm emission
resulting from the antenna effect of the PPE was measured
by the comparison between the perylene emission intensity
obtained with excitation of the PPE backbone at 410 nm (IAD
)
and the emission intensity observed by direct excitation of the
perylene centers at 519 nm (IA) (see Table 1).
Table 1. ET Efficiency of Non-Cross Linked PPE-NB
Nanoparticles in Water at Different Perylene Loadings (mol
%), Quantum Yields (Calculated against Coumarin 6), and
Hydrodynamic Diameter Measured by DLS
Dh(DLS)/
% dye % dye emission IAD/I amplification IAD/IA
Φf
nm
Figure 2. Molecular structures of block copolymers: (a) PPE-NB; (b)
BCB-triblock PPE-NHS; (c) ABCBA-pentablock PPE-NHS-TEG.
0.5
3
79
93
97
91-fold
20-fold
24-fold
0.21
0.18
0.12
140 10
80 10
5
132
1
with an excess of norbornadiene in the presence of formic acid,
which acts as a hydride donor.14 The resultant PPE-NB allows
for these materials to react with olefin metathesis catalysts.
Thus, PPE-NB can be reacted with living ring-opening olefin
metathesis polymerizations (ROMP) to create BCB-triblock
PPE-NHS and ABCBA-pentablock PPE-NHS-TEG copoly-
mers (Figure 2). All the polymers were characterized by H
NMR and their relative molecular weights were measured
(GPC).
After cross-linking the PPE-NHS nanoparticles with [H]-
Lys-Cys-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser-Lys-[NH2], we ob-
serve a decrease in the emission intensity with quantum yields
up to 10-fold lower than the quantum yields of the parent non-
cross-linked NP suspensions.
Sensing experiments were performed using a double read-out
of the fluorescence emission at the aforementioned two
emission maxima (i.e., 454 and 604 nm). We first investigated
assays in PBS buffered solutions with suspensions of shell cross-
linked NPs fabricated from the BCB-triblock PPE-NHS with a
1
Nanoparticles were fabricated by dropwise precipitation of
diluted THF polymer solutions into distilled water under
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perylene dye loading of 3 mol %. A 3.3-fold increase at the main
chain emission maximum (454 nm) and a 2.3-fold increase at
the perylene emission maximum (604 nm) were observed after
incubation with trypsin (Figure 4a). These first assays proved
Figure 5. (a) Reversible swelling effect in non-cross-linked NP
suspensions of PPE-NB (0.5% dye loading) before (left) and after
(right) addition of THF. The figure shows a red emissive collapsed
NPs suspension (Dh: 200 nm) and a blue emitting swollen NP
suspension (Dh: 450 nm) under irradiation at 365 nm. (b)
Fluorescence spectra of a 3 mL aqueous NP suspension of PPE-NB
(3% dye loading) with increasing amount of THF. IAD/I is represented
in parentheses (λex: 410 nm).
Figure 4. Trypsin sensing assays. Fluorescence spectra of shell cross-
linked NP suspensions before (red) and after (blue) incubation with
trypsin for different polymers at a core dye loading of 3 mol %: (a)
PPE-NHS and (b) PPE-NHS-TEG.
the feasibility of the proposed model but improvements were
needed. The main drawback is the large numbers of linkages
that need to be cleaved by the protease to provoke a significant
luminescent response. For this reason, a modification on the
polymer design was made to lower the number of succinimidyl
ester groups (NHS block) without lowering the hydrophobic/
hydrophilic ratio of the polymer. The resulting ABCBA-
pentablock PPE-NHS-TEG copolymer has two polynorbor-
nene blocks with different functionalities: the “B” block
contains succinimidyl esters and the terminal “A” block is
functionalized with tetraethylene glycol (TEG). Because of the
spatial proximity of the reactive succinimidyl esters to the
luminescent core of the NP, this approach allows an effective
quenching of the luminescence of the NPs with a lower cross-
linking degree and therefore a faster cleaving process. The
assays performed with PPE-NHS-TEG at a core perylene dye
loading of 3 mol % under identical experimental conditions
confirmed the merits of this new design and showed a 15-fold
increase at the main chain emission maximum (454 nm) and a
12-fold increase at the perylene emission maximum (604 nm).
To confirm that the changes are the result of the cleavage of
the peptide chains, we performed control experiments wherein
the ABCBA-pentablock PPE-NHS-TEG copolymer was cross-
linked with 1,2-bis(2-aminoethoxy)ethane. In this case,
exposure to trypsin resulted in no changes in the emission
(see the Supporting Information).
The expansion of the particles upon cleaving of the
constraining cross-linking peptide chains by the protease can
be mimicked by the addition of THF to a suspension of non-
cross-linked NPs of PPE-NB. The addition of THF produces a
reversible swelling of the nanoparticles with an increase in
diameter from 200 to 450 nm and a concomitant change of
their emission properties. These changes are visually observable
upon irradiation at 365 nm (Figure 5a). Addition of other water
miscible solvents (acetone, methanol, ethanol) in which the
copolymer is insoluble did not produce a similar effect.
Furthermore, addition of THF to the shell cross-linked NPs
also produced a negligible effect on the size and emission
properties of the NPs, confirming that the cross-links constrain
the particles in a collapsed state. Figure 5b represents the
emission spectra of a 3 mL aqueous suspension of non-cross
linked NPs of PPE-NB with different amounts of THF.
Swelling of the NPs increases the intensity of both the main
chain and dye maxima. Nevertheless, the enhancement of both
maxima is different so the IAD/I ratio decreases as the NPs
swell. This can be explained by considering that swelling with a
good solvent like THF has two opposite effects on the emission
properties. It enhances the total emission intensity by solvating
the chains and reducing interpolymer π−π interactions that
produce self-quenching. However, it also reduces the energy
transfer to the perylenes by separating the polymer chains. We
have previously shown that energy transfer to low energy
emissive sites is enhanced by polymer aggregation.17 The
balance of these opposite effects determines the ratio of both
maxima and explains the observed behavior.
In summary, tunable highly emissive organic NPs have been
developed as selective nanoprobes for protease sensing. The
design takes advantage of the amplification of the fluorescence
emission of the dye by ET of the light harvested by the
backbone of the conjugated polymers and provides a double
read-out of the fluorescence emission. An exquisite tuning of
the fluorescent emission intensity of NPs was performed by
means of aggregation induced quenching by cross-linking using
a peptide as a shell cross-linking agent. Our results suggest that
the polymer cross-linking effectively strains the NP into a more
tightly aggregated and quenched state. The sensory response is
ascribed to a swelling-like (strain-release) mechanism in
response to the protease cleavage of the cross-links, which
results in a “turn-on” fluorescence response. Sensing assays
under physiological conditions provide a 15-fold increase of the
luminescence intensity after incubation with the protease.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details including synthetic procedures, character-
ization of all compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
tswager@mit.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are appreciative of support from the Air Force Office of
Scientific Research (FA9550-10-1-0395). Carlos Cordovilla
recognizes a postdoctoral grant from the MICINN.
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