The PEG-fluorochrome shielding approach for targeted probe design

Article abstract of DOI:10.1021/ja309085b

We provide a new approach for fluorescent probe design termed PEG-fluorochrome shielding , where PEGylation enhances quantum yields while blocking troublesome interactions between fluorochromes and biomolecules. To demonstrate PEG-fluorochrome shielding, fluorochrome-bearing peptide probes were synthesized, three without PEG and three with a 5 kDa PEG functional group. In vitro, PEG blocked the interactions of fluorochrome-labeled peptide probes with each other (absorption spectra, self-quenching) and reduced nonspecific interactions with cells (by FACS). In vivo, PEG blocked interactions with biomolecules that lead to probe retention (by surface fluorescence). Integrin targeting in vivo was obtained as the differential uptake of an ¹¹¹In-labeled, fluorochrome-shielded, integrin-binding RGD probe and a control RAD. Using PEG to block fluorochrome-mediated interactions, rather than synthesizing de novo fluorochromes, can yield new approaches for the design of actively or passively targeted near-infrared fluorescent probes.

Full text of DOI:10.1021/ja309085b

Communication
pubs.acs.org/JACS
The PEG-Fluorochrome Shielding Approach for Targeted Probe
Design
Yanyan Guo,† Hushan Yuan,† William L. Rice,‡ Anand T. N. Kumar,‡ Craig J. Goergen,‡
Kimmo Jokivarsi,‡ and Lee Josephson*,†,‡
‡
†Center for Translational Nuclear Medicine and Molecular Imaging and Martinos Center for Biomedical Imaging, Massachusetts
General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, United States
S
* Supporting Information
through PEG-fluorochrome improvement. PEG has been used
to extend protein circulation and alter surface chemistry,4 but
ABSTRACT: We provide a new approach for fluorescent
probe design termed “PEG-fluorochrome shielding”,
where PEGylation enhances quantum yields while block-
ing troublesome interactions between fluorochromes and
biomolecules. To demonstrate PEG-fluorochrome shield-
ing, fluorochrome-bearing peptide probes were synthe-
sized, three without PEG and three with a 5 kDa PEG
functional group. In vitro, PEG blocked the interactions of
fluorochrome-labeled peptide probes with each other
(absorption spectra, self-quenching) and reduced non-
specific interactions with cells (by FACS). In vivo, PEG
blocked interactions with biomolecules that lead to probe
retention (by surface fluorescence). Integrin targeting in
vivo was obtained as the differential uptake of an 111In-
labeled, fluorochrome-shielded, integrin-binding RGD
probe and a control RAD. Using PEG to block
fluorochrome-mediated interactions, rather than synthesiz-
ing de novo fluorochromes, can yield new approaches for
the design of actively or passively targeted near-infrared
fluorescent probes.
its ability to improve fluorochrome behavior has not been
recognized. To demonstrate that “PEG-fluorochrome shield-
ing” is compatible with molecular targeting in vivo, we show
that a PEGylated, fluorochrome-bearing RGD probe lacks
unwanted fluorochrome-mediated interactions yet maintains
desirable RGD/integrin interactions.
We synthesized PEGylated and un-PEGylated RGD and
RAD probes bearing the CyAL5.5 fluorochrome,5 using the
strategy of attaching a multifunctional reagent module to
integrin-targeted peptides6 (see Figure 1). Here three func-
tional groups (F1 = DOTA, F2 = fluorochrome, F3 = 5 kDa
PEG) were attached to a Lys-Lys-βAla-Lys(N3) peptide
scaffold, and the multifunctional reagent module was then
reacted with a linker-targeting module bearing a RGD- or RAD-
targeting peptide. DOTA allowed radiometal chelation for
biodistribution and/or imaging studies.
Although they differ in just a single methyl group (glycine
versus alanine), RGD and RAD peptides have profoundly
different strengths of interactions with integrins (see ref 7, with
further support provided in Figure 4a). A copper-less “click”
reaction joined the terminal dibenzylcyclooctane (DBCO) of
the linker-targeting vehicle to the azide of the trifunctional
reagent, avoiding the possibility of a copper reaction with the
DOTA. DBCO and azide moieties were found to be highly
stable and readily underwent efficient click reactions when
combined. We term as “PEGylated probes” those bearing the
F3 5 kDa PEG functional group, which consists of 115 PEG
units rather than the short linker-PEG, which consists of just 4
such units. Complete structures of functional groups and
targeting peptide structures are given in the Supporting
Information, Figure S1. The molecular weights, along with a
summary of our results, are provided in Table 1.
We examined the interactions between the 5 kDa PEG and
the CyAL5.5 fluorochrome using the three PEGylated and
three un-PEGylated compounds from Figure 1c, as shown in
Figure 2. In PBS, our three un-PEGylated peptides (5a, 6a, 6b)
exhibited “an aggregation peak” at ∼630 nm, typical of stacked
fluorochromes, while three PEGylated peptides (5b, 7a, 7b)
had reduced aggregation peaks.8 Remarkably, in methanol the
three PEGylated and three un-PEGylated compounds had
ear-infrared (NIR) fluorescent, receptor-targeted pep-
Ntides have been used for pre-operative single photon
emission computerized tomography (SPECT) (or positron
emission tomography, PET) imaging and/or for intra-operative
tumor detection in both pre-clinical1 and clinical settings.2
Though NIR fluorochromes are desirable because of the tissue-
penetrating properties of their light and low background, they
typically involve multiple unsaturated double bonds linking
multiple unsaturated rings. These features lead to self-
quenching fluorochrome/fluorochrome interactions, high non-
specific binding to many cells, unwanted interactions with
proteins and lipids in vivo, and enterohepatic circulation rather
than renal elimination. Efforts to obtain improved NIR
fluorochromes have often followed a “medicinal chemistry”
approach of synthesizing low-molecular-weight fluorochromes
and screening them for desirable properties.3
Here we provide an example where a poly(ethylene glycol)
(PEG) placed on a peptide along with a NIR fluorochrome
blocks troublesome interactions between fluorochromes and
biological molecules, increasing quantum yield and permitting
peptide/target binding. The notion that fluorochromes can be
improved not through fluorochrome redesign but rather by
association with PEG can yield new avenues for probe design
Received: September 12, 2012
Published: November 8, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Communication
Figure 2. Physical properties of six compounds from Figure 1c. (a)
Effects of 5 kDa PEG on probe absorption spectra in PBS and
methanol. (b) Quantum yields. (c) Fluorescent lifetimes. (d) FPLC
chromatograms. (e) Globular protein equivalent volumes of
PEGylated and un-PEGylated probes. For the method of determining
volume, see Figure S5.
Figure 1. Synthesis of PEG-fluorochrome-shielded integrin-targeted
probes. (a) A multifunctional reagent module was first synthesized and
attached to a linker-targeting vehicle module via a copper-less “click”
reaction, to yield a multifunctional probe. (b) Synthesis of the linker-
targeting vehicles bearing RGD or RAD peptides. a: DMF/DiPEA (c)
Synthesis of the multifunctional probes with functional groups of F1 =
DOTA, F2 = CyAL5.5 fluorochrome, and F3 = 5 kDa PEG. Reaction
conditions for a: (1) NH2NH2, DMF, (2) CyAL5.5 acid/PyBOP/
DMF/DiPEA, (3) TFA; b: 5 kDa PEG-NHS, DMSO; c: 5a, RGD-
PEG4-DBCO (3a) or RAD-PEG4-DBCO (3b), DMSO; d: 5 kDa
PEG-NHS. For the need of the linker arm for RGD/integrin
interactions, see Figure S4. Compounds were >90% pure by RP-
HPLC and FPLC (see Figure 2d).
1. Since BT-20 cells express the RGD binding integrins,9 this
cell line was used. With the PEGylation of 5a to obtain 5b, cell
fluorescence dropped from 79.7 to 3.1 au. PEGylation also
reduced the binding of RGD-bearing peptides (6a = 26.7 au to
7a = 9.7 au) and RAD-bearing peptides (6b = 20.4 au to 7b =
3.2 au). However, integrin-specific binding (see inset for values
of 6a, 6b, 7a, and 7b) was not affected by PEGylation (p >
0.05). The binding of the PEGylated RAD probe 7b to cells
(3.2 0.2 au) was similar to that of the PEGylated peptide
lacking both RGD and RAD (5b , 3.1 0.2 au). Therefore, our
PEGylated RAD probe, 7b, is a valid control for our PEGylated
RGD probe, 7a, because of its similar physical properties and
because it does not bind integrins.
identical absorption spectra, and those were characteristic of
unaggregated fluorochromes.
We examined the effects of the F3 5 kDa PEG functional
group on quantum yields (Figure 2b) and fluorescent lifetimes
(Figure 2c) using the six fluorochrome-bearing compounds
from Figure 1c. PEGylation produced significant (p < 0.0001,
see Table S1) increases in quantum yield (Figure 2b, 5a vs 5b,
6a vs 7a, 6b vs 7b). PEGylation also produced significant
increases in fluorescence lifetime (Figures 2c and S3, 5a vs 5b,
6a vs 7a, 6b vs 7b), with p < 0.0001 in all cases. PEG yielded
probes with globular protein equivalent volumes of 20−25 kDa
(5b, 7a, 7b, Figure 2d,e), similar to small proteins. Our three
un-PEGylated probes (5a, 6a, 6b) had volumes <1 kDa. Thus,
PEGylation not only blocked fluorochrome/fluorochrome
interactions but also dramatically increased probe volume. To
determine whether PEG-fluorochrome shielding occurred in
biological systems, we examined the effects of PEGylation on
the binding of probes to cells and on the elimination of probes
after injection.
A measure of the ability of the 5 kDa PEG to block
nonspecific binding in vivo was obtained by probe retention/
elimination after an intramuscular (i.m.) injection (Figure 3c−
f). Mice received dual i.m. injections of 6b (RAD, un-
PEGylated) or 7b (RAD, PEGylated), with time dependence
of surface fluorescence shown in Figure 3c−d. Two regions of
interest (circles near the shoulder) were used to quantify
whole-body surface fluorescence, while a single region of
interest near the bladder was used to quantify bladder
fluorescence and renal elimination. Results for whole-body
and bladder fluorescence are quantified in Figure 3e,f.
PEGylated 7b showed pronounced renal elimination at 4 h
and a lack of whole-body fluorescence. The un-PEGylated 6b
exhibited strong nonspecific interactions, evidenced by the
retention of whole-body fluorescence even at 48 h post
injection.
A summary of the physical properties of our compounds and
their behavior in biological systems in vitro and in vivo is
provided in Table 1. In vitro the F3 5 kDa PEG functional
group reduced the interactions of fluorochromes with each
The ability of the F3 5 kDa PEG to reduce the nonspecific
binding to cultured cells, while maintaining specific binding, is
shown in Figure 3a,b, with numerical values provided in Table
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Journal of the American Chemical Society
Communication
Figure 4. In vivo integrin targeting using the PEG-fluorochrome-
shielded RGD (7a) and RAD (7b) probes. (a) Tissue concentrations
of 111In-labeled 7a and 7b. Numerical data are presented in Table S2.
(b) SPECT-CT image of 7a. Arrows indicate dual BT-20 tumors. Data
are at 24 h post i.v. injection.
provides a method of determining the total RGD/integrin
binding of cells (Figure 3) or tissues (Figure 4a). Integrin-
specific RGD binding to the stomach, spleen, and small
intestine results from a wide range of RGD integrins on diverse
cells and tissues.10 A SPECT/CT image of a 7a-injected animal
is shown in Figure 4b.
Figure 3. Effect of PEG on the specific and nonspecific binding to cells
and on probe elimination. Cell fluorescence (BT-20 cells) after
incubation with the six fluorochrome-bearing peptides from Figure 1c
was determined by FACS as shown in (a), with peak fluorescence
tabulated in (b). (c,d) Whole-body surface fluorescence of mice after
6b (RAD control, un-PEGylated) or 7b (RAD control, PEGylated)
injection at 1 nmol/site. Circles are the regions of interest used to
quantify whole-body (e) or bladder (f) surface fluorescence. (e)
Quantification of whole-body surface fluorescence of seven mice; gray
bars for 6b, black bars for 7b. (f) Quantification of bladder
fluorescence for seven animals.
A model of the PEG-fluorochrome 7a probe, consistent with
our observations and not drawn to scale, is shown in Figure 5.
other (absorption spectra, quantum yield, Figure 2a,b),
increased probe volume (Figure 2e), reduced the nonspecific
interactions with cells (Figure 3a,b), and reduced the
interactions with biomolecules that lead to the retention of
fluorochrome-bearing probes after injection (Figure 3c,d).
To demonstrate that our PEG-fluorochrome shielding
allowed RGD/integrin interactions in vivo, the DOTA func-
tional groups of the PEGylated RGD (7a) and PEGylated RAD
(7b) probes were labeled with 111In and their biodistributions
determined in a BT-20 xenograft system (Figure 4a, Table S2).
Specific RGD binding occurred in the tumor, stomach, small
intestine, lung, and heart. The differential binding of RGD and
RAD probes indicates integrin-specific uptake (see above) and
Figure 5. Model of a PEG-fluorochrome-shielded probe (7a) and its
binding to an integrin.
The 5 kDa PEG forms a cloud around the fluorochrome,
blocking nonspecific interactions (Figure 2b,c) but permitting
integrin interactions (Figures 3b and 4a). PEG provides most of
the probe volume (Figure 2e). However, an essential (Figure
S4), short PEG linker separates the integrin-binding RGD
targeting peptide from the PEG-shielded fluorochrome. With
Table 1. Physical Properties and Behavior in Biological Systems of Multifunctional Reagent Modules (5a, 5b) and Un-
PEGylated (6a, 6b) and PEGylated Probes (7a, 7b)
F3 5 kDa
PEG
target
MW
MS obsd
(Da)
quantum
b
fluor lifetime
vol by FPLC
cell fluor
c
d
e
f
compd
5a
peptide
(Da)
yield
(ns)
(kDa)
(au)
i.m. inject.
no
none
none
RGD
RAD
RGD
RAD
none
1667.0
6805.1
2848.3
2862.4
7987.4
8001.4
800.01
1666.2
0.061 0.006
0.31 0.04
0.66 0.02
0.52 0.05
0.50 0.02
0.81 0.02
0.80 0.03
0.40 0.03
0.4
20
0.4
0.4
25
25
79.7 4.3
3.1 0.2
26.7 0.6
20.4 0.4
9.7 0.3
3.2 0.2
n.o
a
5b
yes
6850 (peak) 0.17 0.008
6a
no
no
2848.3
2862.2
0.082 0.004
0.081 0.004
6b
retained
7a
yes
yes
no
7980 (peak) 0.20 0.01
8000 (peak) 0.20 0.01
7b
eliminated
g
CyAL5.5
799.31
0.08 0.004
n.o.
a
b
c
d
e
f
g
Bold = 5 kDa PEG. Figure 2b. Figure 2c. Figure 2e. Figure 3b. Figure 3c,d. n.o. = not observed.
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Journal of the American Chemical Society
Communication
Hilderbrand, S. A.; Shao, F.; Josephson, L. J. Comb. Chem. 2010, 12,
57.
this design, PEG shielded the fluorochrome and blocked
fluorochrome-mediated interactions but permitted RGD/
integrin interactions.
(7) (a) Mulder, W. J.; Strijkers, G. J.; Habets, J. W.; Bleeker, E. J.; van
der Schaft, D. W.; Storm, G.; Koning, G. A.; Griffioen, A. W.; Nicolay,
K. FASEB J. 2005, 19, 2008. (b) Kok, R. J.; Schraa, A. J.; Bos, E. J.;
Moorlag, H. E.; Asgeirsdottir, S. A.; Everts, M.; Meijer, D. K.; Molema,
G. Bioconjugate Chem. 2002, 13, 128.
(8) Galande, A. K.; Hilderbrand, S. A.; Weissleder, R.; Tung, C. H. J.
Med. Chem. 2006, 49, 4715.
(9) (a) Montet, X.; Funovics, M.; Montet-Abou, K.; Weissleder, R.;
Josephson, L. J. Med. Chem. 2006, 49, 6087. (b) Montet, X.; Montet-
Abou, K.; Reynolds, F.; Weissleder, R.; Josephson, L. Neoplasia 2006,
8, 214.
The ability to improve fluorochromes using covalent linkages
between fluorochromes and PEGs, rather than by de novo
fluorochrome synthesis, can yield many new avenues for the
design of actively or passively targeted fluorescent probes.
When the goal is a receptor binding (actively targeted) probe,
the strategy of attaching a PEG and a fluorochrome to a
common amino acid or peptide can be employed. Our version
of this general strategy employed a modular synthetic route
(Figure 1a) using a trifunctional reagent module [(DOTA)-
Lys(CyAL5.5)-Lys(5 kDa PEG)-βAla-Lys(N3), or 5b]. The
amino group of an RGD-targeting peptide was reacted with a
short PEG linker featuring a terminal DBCO group, and a
copper-less click reaction joined the reagent module and linker
RGD-targeting peptide. For the many intra-operative fluo-
rescent imaging applications,11 passively targeted (untargeted),
PEG-shielded fluorochromes with optical properties similar to
ICG, but which have lost their troublesome interactions with
biological molecules, might be employed.
(10) Humphries, J. D.; Byron, A.; Humphries, M. J. J. Cell. Sci. 2006,
119, 3901.
(11) Sevick-Muraca, E. M. Annu. Rev. Med. 2012, 63, 217.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed synthesis of all compounds, methods for cell binding
assay, animal model, surface fluorescence imaging, absorption
spectra, quantum yield, volume determination, SPECT-CT,
fluorescence lifetime, biodistribution, and additional data,
including expanded structure of compounds, more absorption
and emission spectra, fluorescence lifetime curve, and displace-
ment by direct PEG probe. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
ljosephson@mgh.harvard.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by R01’s from the NIH: EB 011996
and EB 009691.
■
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