Probing two PESIN-indocyanine-dye-conjugates: Significance of the used fluorophore

Article abstract of DOI:10.1039/c9tb01794a

Peptide-dye-conjugates hold a great promise in application for biological and medical imaging of cellular processes and in delineation and characterization of human tumors. In particular, indocyanine dyes are of great interest due to their reported superior properties such as absorption and emission in the near-infrared (NIR) spectral range, favorable Stokes shifts and their well-studied safety profile in humans. In this study, we investigated and describe the influence of indocyanine dyes on different properties of the final peptide-dye-conjugates. As a target peptide, PESIN, a bombesin derivative, was used as a model peptide which addresses GRP receptors overexpressed on different malignancies. Here, we map similarities and differences of the fluorescent conjugates and by this elucidate the influence of the dyes on different properties of the formed conjugates. We performed the dye syntheses, subsequent bioconjugation reactions and in the following investigated the optical properties, water/octanol distribution coefficients and target receptor affinities by in vitro competitive binding studies on PC-3 cells. The obtained results give a handrail to medical and biological researchers planning studies involving indocyanine dye biomolecule conjugates.

Full text of DOI:10.1039/c9tb01794a

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Materials Chemistry B
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Probing two PESIN-indocyanine-dye-conjugates:
significance of the used fluorophore†
Cite this:DOI: 10.1039/c9tb01794a
a
b
c
c
b
Ralph Hubner, * Vanessa Benkert, Xia Cheng, Bjorn Wangler, Roland Kramer
¨
¨
¨
¨
a
and Carmen Wangler
*
¨
Peptide-dye-conjugates hold a great promise in application for biological and medical imaging of
cellular processes and in delineation and characterization of human tumors. In particular, indocyanine
dyes are of great interest due to their reported superior properties such as absorption and emission in
the near-infrared (NIR) spectral range, favorable Stokes shifts and their well-studied safety profile in
humans. In this study, we investigated and describe the influence of indocyanine dyes on different
properties of the final peptide-dye-conjugates. As a target peptide, PESIN, a bombesin derivative, was
used as a model peptide which addresses GRP receptors overexpressed on different malignancies. Here,
we map similarities and differences of the fluorescent conjugates and by this elucidate the influence of
the dyes on different properties of the formed conjugates. We performed the dye syntheses, subsequent
bioconjugation reactions and in the following investigated the optical properties, water/octanol
distribution coefficients and target receptor affinities by in vitro competitive binding studies on PC-3
cells. The obtained results give a handrail to medical and biological researchers planning studies
involving indocyanine dye biomolecule conjugates.
Received 21st August 2019,
Accepted 9th January 2020
DOI: 10.1039/c9tb01794a
rsc.li/materials-b
and a disproportionately high plasma half-life. Other modifications
with small molecule derivatives of the heptamethine dye family
1
. Introduction
3,4
Indocyanine green (ICG) (Fig. 1) is the most prominent member lead, for example, to cypate-2-deoxy-D-glucose (Cypate-2-DG) which
18
of the polymethine dye family. ICG has an odd number of seven mimics [ F]FDG, the gold standard in molecular imaging for
methine groups with alternating single and double bonds clinical diagnostics via positron emission tomography (PET),
forming a large conjugated p-system. In particular, the hepta- however omitting ionizing radiation. Many different studies
methine ICG is a prominent member of this fluorophore class in concerning polymethine-based dyes are available, in particular
2
,5
imaging applications because of its low toxicity, if not irradiated using penta-(Cy5) and heptamethine (Cy7) backbones,
with UV-light, and weak side effects. Advanced optical properties biomedical application (particularly in cancer research).
for
1
–14
such as absorption and emission in the near-infrared (NIR) The indocyanine green molecule has undergone a chemical
spectral range and biodistribution properties different from other evolution over the past 20 years. Different functional groups
dye molecules have made this compound ideal for medical were introduced into the molecule, e.g., carboxylic acids for
diagnostics in the last 60 years. Different derivatives of ICG have further conjugation, sulfonic acids for increased water solubility,
been investigated and reported in terms of their biomedical less or more aromatic systems on the periphery of the molecule for
0
applications. One example is 1,1 bis-(4-sulfobutyl)indotricarbo- increased lipophilicity and tailored fluorescence properties. Even
0
cyanine-5,5 dicarboxylic acid diglucamide monosodium salt the polymethine backbone experienced a ‘‘chemical’’ upgrade by
1
,2
(
SIDAG) (Fig. 1), which exhibits an enhanced hydrophilicity introducing a 1-chloro-cyclohex-1-ene moiety into the center of the
15
molecule to increase its stability towards UV-light. Furthermore,
this functionalization provides the opportunity of introducing
other molecular structures. IR-820 (Fig. 1) is one of the most
prominent representatives of this group of agents. Interestingly, in
animal models, it was shown that derivatives of IR-820 accumulated
in apparently any solid tumor. This tumor-seeking characteristic is
attributed to hypoxia-triggered overexpression of organic anion
a
Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine,
Medical Faculty Mannheim of Heidelberg University, Theodor-Kutzer-Ufer 1-3,
6
8167 Mannheim, Germany. E-mail: ralph.huebner@medma.uni-heidelberg.de,
carmen.waengler@medma.uni-heidelberg.de
b
c
Institute of Inorganic Chemistry, Heidelberg University, Im Neuenheimer Feld 274,
6
9120 Heidelberg, Germany
Molecular Imaging and Radiochemistry, Department of Clinical Radiology and
Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University,
Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany
8
transport proteins. Approaches to increase target-specificity of dye
molecules to target tissues comprised the conjugation to small
1
6–19
1–4
20,21
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c9tb01794a molecules
such as carbohydrates
or other drugs,
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Fig. 1 Structures of Indocyanine green (ICG), SIDAG and IR-820.
7
,12,15,22–27
3,28–31
small linear peptides,
macromolecules
peptidic macrocycles,
3
2,33
13,34
or even antibodies.
The use of NIR-fluorescent dyes, namely heptamethine
derivatives, is beneficial in terms of medical diagnostics because
a potentially disturbing autofluorescence of the tissue does not
superimpose fluorescence in the NIR-range. Therefore, a pene-
tration depth of several millimeters up to centimeters was
2
,35
Fig. 2 Structures of the ICG-derivatives LS277 and CK002 used in this
study for bioconjugation. Both contain only one carboxylic functionality
for conjugation, but are symmetric (LS277) or asymmetric (CK002) and
comprise different numbers of hydrophilic functional groups and annulated
proposed for the emitted light of the NIR dyes.
the detection sensitivity of fluorescent dye markers
proposed to be high thus requiring low amounts of the applied
agent for successful imaging. Applications for pre-operative rings.
Additionally,
3
6,37
was
9
,38
imaging of malignant melanoma
were discussed extensively,
3
,10,11,14,32,39–41
with special focus on image-guided surgery
together
optically guided robotic supported annulated aromatic rings, only two sulfonic acid groups and
6,42,43
with lymph node mapping,
43,44
45,46
biopsy
and even photo-theranostic approaches.
high steric demand. In contrast, CK002 is a highly hydrophilic
Considerable work, including experiments on xenograft dye molecule with three sulfonic acid groups and low steric
1
,4,12,23,25,28–30
mice models, was conducted
of ICG derivative-peptide-conjugates towards cancer cells and aimed to see the influence of these two different dye molecules
tumors could be proven by in vitro and in vivo experiments and on the ready bioconjugates with PESIN (PEG -BBN7-14). As the
no loss of the fluorescence properties of the conjugated dyes was bioconjugates – just as the conjugated polymethine dyes – can
where target specificity demand and an end-on carboxylic acid group. In this study, we
3
3,24,26,27
observed during these experiments.
be expected to represent two end points of a distribution of
What is still missing is a direct comparison of different compounds with different properties, we thus intended to
ICG-derivatives in terms of their influence on the properties of determine if the actually used polymethine significantly influences
the formed peptide-dye-conjugates. Especially with regards to the properties of peptidic conjugates regarding chemical, physical
costly multi-step synthesis of demanding dye molecules – versus and biological properties. Thereby, we will provide guidance with
using inexpensive commercially available dyes – it is not clear respect to the huge available library of polymethine dye molecules,
whether the use of a more elaborate fluorescent ICG dye brings in choosing the best-suited dye for peptidic conjugation.
significant advantages in terms of chemical, physical or biological
Our first objective was to use IR-820 without further modification
properties of the target-specific peptide-dye-conjugate and thus if for conjugation by substitution of the chlorine atom in the central
the use of an optimized dye is worth the additional synthetic effort. position by a terminal primary amine of a peptidic structure to form
Hence, in this study, we endeavored to answer this question and the peptide-dye-conjugate structure. We however observed that the
report the direct effects of using different ICG-derivatives on formed product was not stable towards further nucleophilic attacks
47,48
the chemical, physical and biological properties of the final (Fig. 3A). This lability was also reported by other groups.
There-
peptide-dye-conjugates.
after, we decided to modify the starting compound IR-820 via a
one-step synthesis to obtain a carboxy-modified derivative being
applicable for further conjugation by stable acid-amide coupling.
Dye material LS277 could be obtained by a palladium-catalyzed
Suzuki cross-coupling between the meso-chloride IR-820 and
2. Results and discussion
1
5
For this purpose, we chose two different ICG-derivatives as model 4-carboxyphenylboronic acid in water (Fig. 3B). The formed
compounds (Fig. 2). Dye LS277 is derived from IR-820, a com- C–C-bond was found to be stable in subsequent experiments.
mercially available and inexpensive compound (often termed as This reaction is furthermore feasible in gram quantities and
new indocyanine green) being thus easily accessible in even large the product can be obtained by simple recrystallization from
amounts. The second model ICG-based dye used was CK002 methanol/water.
offering several other advantages (vide infra). These two dyes were
As a second model ICG-based dye exhibiting elaborate physical
chosen because they span a wide range of properties. On the properties, we applied the asymmetrically substituted derivative
17,18,27
one hand, LS277 is a highly hydrophobic dye molecule with CK002 (Fig. 2 and 4).
Dye CK002 comprises three sulfonic
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Fig. 3 Schematic depiction of the reaction of IR-820 with a peptide amine and following side reaction with another nucleophile (A) and synthesis of
LS277 from IR-820 (B). (i) DIPEA + NH -R, 1–2 h; (ii) H O, [Pd(PPh ], 4-carboxyphenylboronic acid, 6–8 h.
2
2
3 4
)
acid groups for increased water solubility accompanied by a (Fig. 4) is however complex as the condensation to the meso-
19
decreased lipophilicity as well as one carboxylic acid functionality chloride is a synthetic step which must be conducted under
on one end of the molecule, which can be used for further strictly inert conditions. The last synthetic step was again a Suzuki
conjugation. The use of such an asymmetric dye comprising coupling, which offers the possibility to introduce different
only one carboxylic acid exhibits the advantage of avoiding side substituents into the dye. The methine color center can be
reactions such as the formation of dimeric peptide dye con- tailored to optimize and fine-tune the absorption maximum
1
8
jugates (the formation of such side products was observed using (lmax), Stokes shift or quantum yields.
dye molecules comprising two carboxy acid groups, at least in To exploit the influence of the respective fluorescent dye on
solid phase-supported synthesis). The synthesis of this molecule a peptidic bioconjugate, we chose PESIN (PEG -BBN7-14),
3
Fig. 4 Schematic depiction of the synthesis pathway towards CK002. (i) 3-Methyl-2-butan-one, NaOAc, CH
butansultone, 1,2-dichlorbenzene, 180 1C, 8 h; (ii) (b) 1,4-butansultone, 1,2-dichlorbenzene, 110 1C, 12 h; (iii) POCI , DMF, 0 1C, 30 min; cyclohexanone,
0 1C, 6 h; ice, 0 1C, 5 h; (iv) (a) NaOAc, EtOH, 4 h, 80 1C; (iv) (b) l h, HPLC; (v) H 0, [Pd(PPh ], phenylboronic acid, 6–8 h, HPLC.
3
COOH, 130 1C, 2 h; (ii) (a) 1,4-
3
5
2
3 4
)
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a Bombesin (BBN) derivative, as a model peptide. PESIN combination with radioisotopes for Positron Emission Tomography
specifically addresses the Gastrin-Releasing Peptide Receptor (PET) or Single Photon Emission Computed Tomography (SPECT)
(
GRPR) which is overexpressed in different human malignancies. imaging. However, fluorescent dye conjugates of BBN analogs were
3,4,12,16,31,50
Bombesin is a 14-mer peptide that binds with high affinity and also described.
specificity to the GRPR. These G protein-coupled receptors have The two target bioconjugates could be obtained by straight-
different physiologic functions in the gastrointestinal tract and forward chemical synthesis (Fig. 5). The PESIN peptide was
nervous system under regular circumstances. An overexpression of obtained via solid phase-supported synthesis using a standard
5
1
this receptor was demonstrated in several cancers including Fmoc-based protocol. The linker structure as well as the dyes
3
5,49
prostate, breast, pancreas and gastrointestinal tumors.
Based on the pharmacophoric amino acid sequence BBN7-14
LS277 and CK002 were conjugated to PESIN on the solid
,
support at elevated temperatures to obtain the target-specific
various analogues of BBN were tested as imaging probes, mostly in PESIN-dye-conjugates (PDC) PDC 1 and PDC 2, respectively.
Fig. 5 (a) Schematic depiction of the synthesis pathway towards the PESIN-dye-conjugates PDC 1 and PDC 2. (i) Swelling 45 min; piperine/DMF (1 : 1),
5
3
3
min; 4 eq. activated amino acid for 30 min (activation: 3.9 eq. HBTU, 4 eq. DIPEA, 2 min); (ii) piperine/DMF (1 : 1), 5 min; 4 eq. activated amino acid for
0 min (activation: 3.9 eq. HBTU, 4 eq. DIPEA, 2 min); (iii) piperine/DMF (1 : 1), 5 min;4 eq. activated Fmoc-NH-PEG -COOH for 30 min (activation:
.9 eq. HBTU, 4 eq. DIPEA, 2 min); (iv) 1.5 eq. activated dye for 3 h at 80 1C (activation: 1.425 eq. HBTU, 1.5 eq. DIPEA, 10 min); (v) TFA/TIS (95 : 5 v/v), 1 h,
3
HPLC. (b) Schematic depiction of PDC 1 and PDC 2.
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Table 1 Summary of the chemical, physical and biological properties of the PESIN-dye-conjugates PDC 1 and PDC 2 compared to the unconjugated
18
dyes, LS27752 and CK002
a
a
Parameter
PDC 1
LS277
PDC 2
CK002
log D
Not determinable
801 nm
4.78 M cm
813 nm
12 nm
0.036
32.22 ꢁ 0.72 nM
ꢀ1.03 ꢁ 0.11
ꢀ4.00 ꢁ 0.05
ꢀ4.00 ꢁ 0.05
l
max (Abs)
804 nm
766 nm
761 nm
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
log e
5.00 M cm
5.17 M cm
5.44 M cm
l
max (Em)
815 nm
11 nm
0.003
—
784 nm
18 nm
0.114
20.58 ꢁ 0.55 nM
781 nm
20 nm
0.090
—
Stokes shift
j
IC50
f
Quantum Yield
*
a
*
IC50 BBN: 8.07 ꢁ 0.56 nM. Optical properties taken from ref. 18 and 52, respectively.
Attempts to conjugate the dyes at ambient temperature, even change of absorption properties or a loss of fluorescent char-
using prolonged reaction times, did not result in the formation acteristics (Table 1). In contrast, a significant, 10-fold increase
of the target products. Even at elevated temperature conditions, of the fluorescence quantum yields of PDC 1 compared to LS277
the observed yields were rather low for both PDCs ranging was observed. This can be attributed to a considerably reduced
between 8 and 10%. An optimization of the reaction pathway aggregation behavior of PDC 1 (Fig. 6) compared to LS277 and
might be achievable by conjugation of dyes and PESIN in as a consequence, a lowered energy-transfer. Self-aggregation
solution to increase the conjugation efficiency of the rather induces a significant energy transfer between molecular units
bulky fluorescent dye systems.
and leads to intense fluorescence quenching. In the peptide dye
As we aimed to determine the influence of the respective conjugate, such self-aggregation and its disturbing intermolecular
ICG dye derivative on the chemical, physical and biological energy-transfer is hindered, which results in higher fluorescence
53–55
properties of their bioconjugates, we first examined the octanol/ quantum yields.
The same trend can be observed in the CK002/
water partition coefficient of the dyes and their PDCs. This PDC 2 system although the effect of increased quantum yields upon
coefficient, usually given as logD, gives a good indication of the dye conjugation is less pronounced for PDC 2 (Table 1). Thus, the
possible biodistribution of bioactive molecules and their con- peptide conjugation did not result in adverse effects on absorption
jugates in vivo and is therefore an important parameter for or fluorescent properties of the dyes, but even improved fluorescent
potential imaging agents. Water soluble (hydrophilic) molecules quantum yields, enabling a good prognosis of these characteristics
are usually cleared by the kidneys while lipophilic compounds upon dye conjugation.
1
are segregated by the liver. For dye LS277, a log D of ꢀ1.03 ꢁ
Finally, we also intended to determine the influence of the
0
.11 was found. For dye CK002, virtually no accumulation was respective ICG dye on the biological properties of the formed
found in the octanol phase (log D = ꢀ4.00 ꢁ 0.05). The con- bioconjugates. For this purpose, the IC50 values of PDC 1 and
siderably higher hydrophilicity of CK002 compared to LS277 is PDC 2 towards the target GRP receptor were determined on
not surprising since sulfonic acid groups increase water solubility, human prostate carcinoma PC-3 cells using bombesin (BBN) as
1
25
4
whereas annulated aromatic rings increase lipophilicity. The a reference compound and [ I]-Tyr -bombesin as a competitor
influence of the sulfonic acid groups is retained upon bio- (Fig. 7). The IC50 values were determined to be 8.07 ꢁ 0.56 nM,
conjugation as the same log D value of ꢀ4.00 ꢁ 0.05 was found
for PDC 2. In contrast, the log D was not determinable for PDC 1
as the compound was not soluble in either solvent to a significant
extent, but formed a layer between both the phases. Possible
explanations might be the formation of aggregates or an alignment
of the molecules at the phase boundary along their default
preferences. This behavior however could be challenging
regarding an in vivo application of the bioconjugate resulting
in limited target accessibility due to unspecific interactions, but
could as well be unproblematic as the substance is used in high
dilution during in vivo imaging. These results indicate that the
used dye can have a significant influence on the hydrophilicity
of the target bioconjugate.
In the following, we also determined the physical characteristics
of the fluorescent PDCs in solution. At first, absorption and
emission characteristics were determined. We observed only a
slight shift of the absorption maximum for both compounds
Fig. 6 Absorption spectra of PDC 1 (top left) and PDC 2 (bottom left) in
PBS and corresponding emission spectra of PDC 1 and PDC 2 on the right
compared to the unconjugated dyes. The same was true for
1
8,52
Stokes shifts and absorption coefficients.
Functionalization
(
excitation wavelength lex = 705 nm). Quantum yields were determined
f
of the dyes with the PESIN molecule thus did not lead to a relative to ICG (j = 0.13 in DMSO).
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data suggest a general suitability of both dyes to form bio-
conjugates with mostly retained binding characteristics.
3
. Conclusions
The results of the performed study reveal that for the development
of ICG-based near-infrared fluorescent peptide-dye-conjugates, the
choice of the dye is of minor significance in terms of the properties
of the resulting conjugates (Table 1).
To arrive at this conclusion, we directly compared two rather
similar PESIN-dye-conjugates, differing only in the used ICG-
based fluorescent dye: One conjugate bearing the rather easily
chemically accessible dye LS277 (exhibiting annulated benzene
Fig. 7 Results of the competitive displacement assays of PESIN-dye-
conjugates PDC 1 and PDC 2 on human prostate carcinoma PC-3 cells rings and two sulfuric acid groups and a central functional
125
4
using [ I]-Tyr -bombesin as competitors. All the experiments were group for further functionalization) and the other comprising
performed thrice, each experiment in triplicate. The IC50 values for BBN
dye CK002, which has to be synthesized from scratch via a time-
(
being used as the reference compound), PDC 1 and PDC 2 were
consuming multistep synthesis (exhibiting three sulfonic acid
groups for increased water solubility and one functionalization
motif at one sterically easily accessible peripheral position of
the molecule).
determined to be 8.07 ꢁ 0.56 nM, 32.22 ꢁ 0.72 nM and 20.58 ꢁ 0.55 nM,
respectively.
3
2.22 ꢁ 0.55 nM and 20.58 ꢁ 0.72 nM for BBN, PDC 1 and
The determination of the chemical, physical and biological
PDC 2, respectively, showing only a slight decrease in receptor properties of the conjugates revealed that: (i) as expected, the
affinity of the peptide upon dye conjugation. This effect was additional sulfonic acid group and the absent annulated rings
somewhat more pronounced in the case of dye LS277 (factor 4 of dye CK002 have an effect on the hydrophilicity of the peptide-dye-
lower target receptor affinity) than for dye CK002 (decrease by conjugate and thus should be able to influence the biodistribution
factor 2.6).
of the bioconjugate, (ii) the photo-physical properties of the dyes
Previous studies with the cyclic RGD peptide c(RGDyK), remain mostly unaltered upon peptide conjugation or even profit
2
8–30
targeting integrin a b ,
somatostatin, targeting somatostatin from conjugation to a peptidic biomolecule by increase of the
analogs functionalized with different fluorescent quantum yields of both conjugates compared to the
v
3
5
,24
receptors and bombesin
fluorescent dyes revealed in some cases a high sensitivity of respective dyes thus not per se favoring one specific dye for
receptor binding towards dye conjugation (especially in the bioconjugation, and (iii) dye CK002 – most probably due to its
5
case of somatostatin–fluorescein-derivatives, depending on more peripheral functionalization and thus conjugation, resulting
the position where the dye was introduced into the peptide in a lower spatial demand of the dye within the bioconjugate –
sequence and the nature of the linker used). In another study, has a somewhat less pronounced effect on the binding affinity
reporting an ICG–bombesin derivative, an even higher receptor of the conjugated peptide compared to dye LS277.
5
binding affinity than for the parent peptide was found. No
Taken together, a specific ICG-based dye can have an effect
large influence of dye conjugation on GRP receptor binding was on the resulting chemical and biological properties of its bio-
5
observed for bombesin–fluorescein-derivatives, and only a conjugates. However, the influence is rather small and so the
slightly decreased binding affinity of the dye conjugates com- significance of the choice of the used dye is limited. Thus, when
pared to the parent compound was found, being in accordance intending to synthesize a peptidic optically active imaging
with the results found here.
agent with tailored chemical, photophysical and biological
It cannot be fully answered if the observed aggregation of characteristics, the used polymethine dye has only a minor
PDC 1 affects its GRPR binding properties. However, at the significance, at least on monomeric PESIN-dye-conjugates under
concentrations used in this assay, the aggregation should be investigation.
negligible (as can also be deduced from the UV-Vis spectra), so
that it can be assumed that if there is an influence, it is very
small. We thus suppose that the higher target receptor affinity
Conflicts of interest
of PDC 2 might be a consequence of the steric arrangement of There are no conflicts to declare.
dye CK002 in conjugate PDC 2. The attachment of PESIN to
LS277 in the center of the dye molecule might lead to a higher
spatial demand and thus a potentially more significant inter-
Acknowledgements
ference of the LS277 dye during the peptide–receptor- The authors thank the German Research Foundation for financial
interaction. In contrast, CK002 is connected to the peptide support (grant number WA3555/5-1), Trina Irico for proofreading of
via a terminal functional group, which might reduce its spatial the manuscript, Dr. Werner Spahl (LMU Munich) for performing
demand, less disturbing the peptide–receptor interaction of the ESI measurements and Tobias Timmermann (IPMB Heidelberg) for
1
conjugate. Nevertheless, the obtained receptor binding affinity performing H-NMR measurements.
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