Near-Infrared Heptamethine Cyanine Dyes for Nanoparticle-Based Photoacoustic Imaging and Photothermal Therapy

Article abstract of DOI:10.1021/acs.jmedchem.1c00771

We have synthesized and characterized a library of near-infrared (NIR) heptamethine cyanine dyes for biomedical application as photoacoustic imaging and photothermal agents. These hydrophobic dyes were incorporated into a polymer-based nanoparticle system to provide aqueous solubility and protection of the photophysical properties of each dye scaffold. Among those heptamethine cyanine dyes analyzed, 13 compounds within the nontoxic polymeric nanoparticles have been selected to exemplify structural relationships in terms of photostability, photoacoustic imaging, and photothermal behavior within the NIR (～650-850 nm) spectral region. The most contributing structural features observed in our dye design include hydrophobicity, rotatable bonds, heavy atom effects, and stability of the central cyclohexene ring within the dye core. The NIR agents developed within this project serve to elicit a structure-function relationship with emphasis on their photoacoustic and photothermal characteristics aiming at producing customizable NIR photoacoustic and photothermal tools for clinical use.

Full text of DOI:10.1021/acs.jmedchem.1c00771

pubs.acs.org/jmc
Article
Near-Infrared Heptamethine Cyanine Dyes for Nanoparticle-Based
Photoacoustic Imaging and Photothermal Therapy
Anna St. Lorenz,∥ Emmanuel Ramsey Buabeng,∥ Oleh Taratula, Olena Taratula,* and Maged Henary*
Cite This: J. Med. Chem. 2021, 64, 8798−8805
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: We have synthesized and characterized a library of
near-infrared (NIR) heptamethine cyanine dyes for biomedical
application as photoacoustic imaging and photothermal agents.
These hydrophobic dyes were incorporated into a polymer-based
nanoparticle system to provide aqueous solubility and protection of
the photophysical properties of each dye scaffold. Among those
heptamethine cyanine dyes analyzed, 13 compounds within the
nontoxic polymeric nanoparticles have been selected to exemplify
structural relationships in terms of photostability, photoacoustic
imaging, and photothermal behavior within the NIR (∼650−850
nm) spectral region. The most contributing structural features
observed in our dye design include hydrophobicity, rotatable bonds,
heavy atom effects, and stability of the central cyclohexene ring within the dye core. The NIR agents developed within this project
serve to elicit a structure−function relationship with emphasis on their photoacoustic and photothermal characteristics aiming at
producing customizable NIR photoacoustic and photothermal tools for clinical use.
organ imaging. In PAI, light absorbed by molecules is released
as heat that causes thermal expansion of the surrounding area
and produces acoustic waves detectable via an ultrasound
probe. The use of exogenous light-responsive PAI agents,
which target tissue of interest, not only allows for tissue-
specific imaging but also offers a possibility for photothermal
therapy (PTT) for the precise removal of diseased tissue. In
efforts to optimize NIR contrast agents for PAI applications,
cyanine dyes have been attractive in general due to their
narrow absorption bands and high molar extinction coef-
ficients.8,9 This is because the properties of heptamethine
cyanine dyes are versatile, which arises from the fact that
modifications to the polymethine chain as well as heterocyclic
moieties allow for tuning of their photophysical features. To
date, heptamethine cyanine dyes (e.g., NIR-I fluorophores
IRDye800CW and FDA-approved indocyanine green
(ICG))10 have shown great potential in superficial biomedical
imaging, serving as fluorescence probes for image-guided
cancer surgery,11,12 gastrointestinal tract imaging,13 and
imaging of various tissues such as cartilages, bone, thyroid
and parathyroid glands, and lymph nodes.14−19
INTRODUCTION
■
Accurate and sensitive medical imaging is a crucial aspect of
earlier diagnosis, offering to reduce an overall disease burden
and better patient outcomes. Current clinical diagnostic
modalities include magnetic resonance imaging (MRI),
positron emission tomography (PET), single-photon emission
computed tomography (SPECT), ultrasound imaging (USI),
X-ray imaging, and fluorescence imaging.1−3 USI has explicitly
been lauded as a favorable approach due to its non-ionizing
and deep tissue-penetrating acoustic signal that relies on the
differences in tissue density of endogenous markers. USI is
widely implemented already for point-of-care diagnostics and is
relatively cost-effective, but improvements in resolution are
desired. On the other hand, fluorescence (optical) imaging has
also been notable due to its high resolution and is used across
the spectrum of basic research to clinical studies to acquire
real-time information.4,5 While fluorescent dyes are commonly
used as exogenous probes in optical imaging, they have proven
to be most beneficial in the ∼650−850 nm region (also known
as the NIR-I window) with greater tissue transmission due to
lower light scattering and energy absorption.6 Even then, there
is a significant challenge for optical imaging detection due to
poor tissue penetration as the fluorescence signal is still only
detectable at shallow depths of <8 mm within the entire NIR-I-
to-NIR-II window (650−1700 nm).7
Received: April 28, 2021
Published: June 3, 2021
Combining features of USI and optical imaging techniques
resulted in an optoacoustic technique, also known as
photoacoustic imaging (PAI), that provides higher resolution
and improved optical contrast for deeper-seated tissue and
https://doi.org/10.1021/acs.jmedchem.1c00771
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 8798−8805
8798

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Heptamethine cyanine dyes investigated here consist
primarily of a two-terminal heterocyclic ring, which is
connected via a polymethine chain, as shown in Figure 1.
Their absorption bands span within the 750−850 nm NIR
region, depending on the chain length and the substitution
patterns on the dye.20−23
As depicted in Scheme 1, various phenylhydrazines 1 were
treated with 3-methyl-2-butanone in acetic acid (HOAc) under
Scheme 1. (A) General Synthesis of Heterocycles 3a−3t;
(B) Substituents R, R1, and R2 in the Molecular Structure of
the Synthesized Compounds 3a−3t
Figure 1. General structure of studied heptamethine cyanine dyes.
On their own, these dyes generally suffer from fast body
clearance, stability, and poor aqueous solubility. While
structural modifications of heptamethine dyes can help
improve these issues, an alternative approach has also been
availed by encapsulating these dyes within nontoxic nano-
particle structures.24 This approach has advantages for
improving solubility in a physiological medium, shielding the
dye molecules and increasing the overall agents’ size, which can
prolong circulation and lower rapid clearance and thus passive
targeting in instances of leaky vasculature (e.g., solid
tumors).24 In this work, we have developed and characterized
a library of newly synthesized dyes to be able to relate key
structural components of the dyes to their photofunctionalities
of the nanoparticle product. Thus, a library of near-infrared
heptamethine dyes has been synthesized; suitable dyes from
the synthesized library were encapsulated within polymeric
nanoparticles and characterized for their photoacoustic and
photothermal capabilities. This study focused on correlating
the chemical structure of heptamethine cyanine dyes with a
variety of distinct functional groups to the functionalities of the
nanoparticle product with the goal to provide a working
knowledge for the development of heptamethine dye-based
nanoparticle probes for PAI-guided photothermal therapy.
RESULTS AND DISCUSSION
■
Synthesis of Heptamethine Cyanine Dyes. To achieve
our goal, heptamethine dyes with open-chain and cyclohexene-
derived polymethine bridges have been designed, synthesized,
and characterized. Structural modifications were performed on
the synthesized heterocycles to study the effect of different
molecular features on the photoacoustic and photothermal
properties of the dyes. Alkyl groups such as methyl, ethyl,
propyl, butyl, and phenyl propyl were introduced at the
nitrogen atom of the heterocycle ring. Likewise, heavy atoms
such as chlorine and bromine atoms were also presented at the
heterocycle ring. These key features were incorporated within
the dyes’ molecular scaffolds to ascertain the effect of electron-
donating groups, rotatable bonds, hydrophobicity, a heavy
atom, and the rigidity of the central cyclohexene ring on the
photoacoustic and photothermal properties.
reflux conditions for 24 h. The resulting crude product was
allowed to cool to room temperature and then neutralized with
sodium bicarbonate. The reaction mixture was then extracted
with dichloromethane, and upon concentration under reduced
pressure, a brown oil of 2,3,3-trimethylindolenine correspond-
ing derivatives 2 was obtained in good yields25 and used
without further purifications.
Compounds 2 were functionalized with different alkylating
agents in acetonitrile (ACN) under reflux conditions for about
12−18 h to afford the heterocyclic salts 3a−3t.26
Open-chain and cyclohexene ring derivatives of heptame-
thine dyes were synthesized according to our reported
procedures.4 As shown in Equation 1, both types of
heptamethine cyanine dyes with an open chain and cyclo-
hexene (n = 0, 2, 3) were prepared.
Heptamethine dyes 4a−4ee were achieved by a condensa-
tion reaction of the heterocycle salts 3a−3f and the
corresponding linker27 in a 2:1 ratio. By heating individual
salts 3a−3f with sodium acetate in acetic anhydride for 2−3 h,
the crude products were produced. The purified products 4a−
The efficiency of most synthetic procedures relies on a
stepwise approach to optimize the overall yield and also to
improve the purity level. We began the synthesis of the
heptamethine dyes by carrying out a Fischer indole cyclization
reaction.
8799
https://doi.org/10.1021/acs.jmedchem.1c00771
J. Med. Chem. 2021, 64, 8798−8805

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Preparation and Characterization of Dye-Loaded
Nanoparticles. Small-molecule dyes have, in general,
demonstrated fast clearance from systemic circulation and
often have no innate specificity to any particular tissue.28,29 To
reduce the clearance rate, provide protection from interactions
and degradation in the blood, and increase the retention within
the tissue of interest (e.g., tumor), synthesized heptamethine
dyes 4a−4ee were encapsulated into a poly(ethylene glycol)-b-
poly(ε-caprolactone) (PEG-PCL) nanoparticle system.29−32
Also, notably, this approach addresses the solubility of the
small hydrophobic molecules for delivery in aqueous solutions
as the PEG-PCL polymer forms a hydrophobic core with a
biocompatible, non-immunogenic, and hydrophilic outer PEG
shell (Figure 2). The herein synthesized dyes (31 in total,
Synthesis of Heptamethine Cyanine Dyes 4a−4ee
4ee were obtained after careful purification via column
chromatography using a 10:1 ratio of DCM/MeOH. The
yields of the synthesized dyes ranged from 15 to 90%.
The synthesized heptamethine cyanine dyes (31 com-
1
pounds) are shown in Table 1, and their corresponding H,
13C NMR, and mass spectra are provided in the Supporting
Information (Figures S1−S55). From Table 1, 18 out of the 31
heptamethine cyanines were designed to contain a six-
membered ring system as part of the methine chain, such as
in compounds 4a−4ee. This ring subsystem confers some level
of rigidity in the synthesized compounds, thereby increasing
the stability of these compounds and minimizing aggregation
in solution. As shown in Table 1, among the 18 heptamethine
compounds synthesized containing a cyclohexene ring, half of
them contain a meso-chlorine atom, while the remaining nine
contain bromine, methyl, or phenyl groups at the meso-
position.
Figure 2. Schematic illustration of the heptamethine cyanine dye
loaded within the PEG-PCL nanoparticle.
Table 1) were encapsulated within the nanoparticle system by
means of a solvent evaporation method and purified via
centrifugation and filtration through a 0.2 μm pore system.33
Heptamethine dye containing a five-membered ring, 4u, was
also synthesized. A cyclopentene moiety within the poly-
methine chain of 4u increases the dye’s rigidity.
Table 1. Substituents R, R1, and R2 in the Molecular Structure of the Synthesized Heptamethine Dye Derivatives 4a−4ee
ID
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
R
R1
R2
R3
R4
n
H
F
Cl
Br
OCH3
F
Cl
H
H
H
H
H
H
H
H
(CH2)3Ph
(CH2)3Ph
(CH2)3Ph
(CH2)3Ph
(CH2)3Ph
(CH2)3Ph
(CH2)3Ph
(CH2)3Ph
CH2CH3
(CH2)3CH3
(CH2)3Ph
(CH2)3CH3
(CH2)3Ph
(CH2)3Ph
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
CH3
H
H
H
H
0
0
0
0
0
3
3
3
0
0
0
3
3
3
3
0
3
0
3
3
2
3
3
3
3
3
3
0
0
3
3
H
Cl
Cl
Cl
H
H
H
Cl
Cl
CH3
Ph
H
H
H
H
H
H
H
OCH3
(CH2CH2)2
(CH2CH2)2
(CH2CH2)2
(CH2CH2)2
(CH2CH2)2
(CH2CH2)2
(CH2CH2)2
(CH2CH2)2
C(CH3)3
(CH2CH2)2
(CH2CH2)2
H
H
H
H
H
H
(CH2CH2)2
(CH2CH2)2
(CH2CH2)2
(CH2CH2)2
Cl
Br
OCH3
H
H
H
H
H
Cl
Br
Br
OCH3
F
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
4s
4
Cl
Ph
Cl
CH3
Ph
CH3
Cl
CH3
Cl
H
C(CH3)3
H
4u
4v
4w
4x
4y
4z
4aa
4bb
4cc
4dd
4ee
CH3
CH3
CH3
CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
H
Ph
Ph
Cl
Br
8800
https://doi.org/10.1021/acs.jmedchem.1c00771
J. Med. Chem. 2021, 64, 8798−8805

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 2. Selected Subset of Heptamethine Cyanine Dyes Loaded within the PEG-PCL Nanoparticles that Have Notably
a
Pronounced Photoacoustic and Heat-Generating (Photothermal) Capabilities
a
(A) Absorbance (nm) and corresponding PA (nm) peaks, arranged in order of increasing wavelength, and (B) absorbance (nm) and temperature
(temperature increase is also reported as ΔT, °C) generated under the 10 min NIR light exposure, arranged in order of increasing heat.
The nanoparticles were analyzed with regard to their size
and population characteristics via dynamic light scattering
(DLS) and were generally found to be between 35 and 95 nm,
with the majority having a PDI of <0.25 and a nearly neutral
charge of 5 mV (Table S1). These are generally regarded as
within the parameters of being able to be used intravenously
for extended blood circulation and leading to more specific
uptake in tumor growths through the enhanced permeability
and retention (EPR) effect.
Evaluation of Photoacoustic and Heat Production
Properties of Dye-Loaded Nanoparticles. Further charac-
terization of the nanoparticles was done by evaluating their
absorption, fluorescence, and photoacoustic (PA) properties
(Figures S56−S58). Most of the studied dyes being loaded
into the PEG-PCL nanoparticle displayed distinguishable PA
signals (within 760−840 nm) that give them credence for PA
imaging (Figure S58). To evaluate the dye-loaded nano-
particles’ capability of heat production under NIR light for
application in photothermal therapy (PTT), the thermal
release was assessed by exposure to an NIR laser light (0.6
W/cm2) that was used to excite the encapsulated dyes for 10
min. While the samples were incubated at 37 °C, the observed
increase in temperature after 10 min under NIR light was 0.5−
21.7 °C (Table S2). Some of these temperatures are high
enough for photothermal therapy (PTT) applications at
temperatures beyond 42 °C and ablative therapy beyond 50
°C (Table 2B).34,35 Of note, water as a control did not produce
heat under the same laser light treatment. The absorbance
peaks of studied encapsulated dyes were recorded pre- and
post-exposure to the 10 min NIR light to evaluate the
photostability of produced formulations for successful
application in PTT (Figure S56).
After evaluation of PAI and heat-generating properties of
nanoparticles loaded with dyes 4a−4ee, a subset of the library
with most distinct PA and heat-generating properties was
identified to establish a relationship between the structure of
the dye and the PAI and PTT functions of the dye-loaded
nanoparticle (Table 2). Certain critical structural features were
identified among the library of tested compounds for their
contribution to PAI and PTT properties. These features
include the nature of the heterocycles, the polymethine chain
within the contrast agents, the presence or absence of a
cyclohexene ring within the dye framework, the presence of a
heavy atom, rotatable bonds, electron-donating groups
(methoxy), and hydrophobicity. Table 2A demonstrates dyes
loaded within PEG-PCL nanoparticles with the most
8801
https://doi.org/10.1021/acs.jmedchem.1c00771
J. Med. Chem. 2021, 64, 8798−8805

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 3. (A) Viability of ES2 ovarian cancer cells after incubation with 4h-loaded nanoparticles (4h-NP) or indocyanine green (ICG) dye for 24 h
at 0.4−11 μM concentrations. Values are shown as a percentage in relation to the untreated cell control. (B) Fluorescence imaging (shown in red,
Ex./Em. 710/775 nm) demonstrating the cellular uptake of 4h-NP or ICG dye after 24 h incubation (5 μM) with included magnified imaging
areas. Nuclei were stained with DAPI (in blue, Ex./Em. 360/460 nm). (C) Ultrasound images overlaid with a PA (unmixed) signal of pelleted ES2
cells (embedded in gel): untreated cells (control) or treated with 4h-NP (10 μM) and ICG (10 μM) for 24 h, depicting 4h (green) and ICG (red).
Scale bar is 1 mm. Insets: temperatures recorded within the cell pellet after 10 min NIR light irradiation (0.6 W/cm2, 808 nm). (D) Normalized PA
spectra generated from images in panel (C) and overlapped with the corresponding spectra of 4h-NP (in water), ICG (in water), and water (as a
control).
pronounced PA signal, depicting PA peaks accompanied by
NIR absorbance peaks arranged in order of increasing
wavelength, whereas the dye-loaded nanoparticles with the
highest heat production under NIR light are listed in Table 2B,
showing a temperate increase along with absorbance peaks.
To confirm the most contributing feature to the PTT
capability of 4g, we evaluated its analog 4h bearing methoxy
groups at the heterocyclic rings under the same experimental
conditions. However, the change in temperature dropped
drastically from 21.7 to 9.8 °C. This suggests that the
substitution of a halogen, in this case, a chlorine atom with a
methoxy group, disfavors the tested dye’s photothermal ability.
As a general trend for the heptamethine cyanine dyes’ scaffolds
with cyclohexene in the middle, only those with halogens
(heavy atom effects), rotatable bonds, and hydrophobicity tend
to give appreciable change in temperature (Table 2B).
We turned our attention to open-chain NIR dyes, and
among the synthesized compounds, only 4i, 4cc, 4p, 4k, and
4d exhibited a detectable change in temperature. As shown in
Table 2B, the order of decreasing change in temperature is 4i >
4cc > 4p > 4k > 4d. In comparison, 4i and 4k are analogs with
an ethyl group and phenyl propyl substituted on the ring
nitrogen. However, as observed in Table 2, 4i with a short
chain tends to give off more than twice the heat than its
counterpart 4k (18 °C vs 7.6 °C, respectively) with a long
chain and an increase in hydrophobicity. In addition, 4cc and
4p are analogs with ethyl and butyl groups substituted on the
ring nitrogen of the indolium, respectively. However, as
observed in Table 2B, 4cc produced a more considerable
change in temperature (17.2 °C) than dye 4p (9 °C). Thus, a
short alkyl chain with halogen-substituted compounds seems
to be more promising as photothermal agents. Changing butyl
to phenyl propyl further decreased the change in temperature
by two units, as observed in Table 2B for dye 4d. Despite the
differences observed in all tested compounds for their
photothermal capabilities, all these compounds were photo-
stable except the control compound 4z, which is a
heptamethine cyanine NIR dye with a cyclohexene poly-
methine chain. As described above, heptamethine cyanine dyes
with cyclohexene in the middle tend to be good photothermal
agents if they exhibit rotatable bonds, hydrophobicity, and
halogens as observed in 4g. However, compound 4z lacks a
rotatable bond off the ring nitrogen , and this might account
for its photoinstability as well as poor thermal output (Δ 0.6
°C). Therefore, based on our observations, indolium and
benzo[e]indolium scaffolds bearing a short alkyl chain in the
ring nitrogen and halogen on the heterocyclic ring with an
open polymethine chain seem to be more favorable as
photothermal agents than their counterpart with a long chain
and higher levels of hydrophobicity. However, the opposite is
true for scaffolds with cyclohexene in the polymethine chain.
8802
https://doi.org/10.1021/acs.jmedchem.1c00771
J. Med. Chem. 2021, 64, 8798−8805

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
In Vitro Assessment of PAI and Photothermal
Behavior of Dye (4h)-Loaded Nanoparticles. To demon-
strate the PA and PTT capabilities of studied heptamethine
cyanine dyes in vitro, the representative nanoparticle
formulation containing 4h dye (4h-NP) was further evaluated
in ovarian cancer cells (ES2) in comparison to the FDA-
approved clinical standard (indocyanine green, ICG). 4h-NP
was selected due to its sharp and distinct PA spectral peak as
well as good heat generation under NIR light exposure (Table
2). To assess the cellular biocompatibility of the developed
imaging nanoparticle, ES2 cells were treated with 4h-NP and
ICG for 24 h, demonstrating no observed toxicity within the
0.4−11 μM concentration range (Figure 3A). Cellular uptake
was confirmed with fluorescence imaging of formulation-
treated ES2 cells, employing intrinsic NIR fluorescence of 4h-
NP and ICG and overlaying with DAPI (nuclei stain), and ES2
cells demonstrated good cellular uptake with substantial
internalization of both 4h-NP and ICG within the cytoplasm
but not crossing to nuclei (Figure 3B), without any visual
indication of toxicity.
molecular features to correlate the photoacoustic and photo-
thermal properties of the final contrast agents encapsulated in
nanoparticles. Key features such as electron-donating groups
(methoxy), rotatable bonds, hydrophobicity, heavy atom
effects, and rigidity of the central cyclohexene ring bearing
halogen at the meso-position were observed based on our
studied compounds 4a−4ee to be the significant contributing
factors to strong photoacoustic signals. However, for photo-
thermal agents, probes with short N-alkyl groups, open-chain
polymethine, and heavy atoms are the underlying features for
good photothermal signals to be generated, while for
cyclohexene-derived polymethine scaffolds, rotatable bonds,
hydrophobicity, and heavy atom effects are essential. As a
proof of concept, our study demonstrated that polymeric
nanoparticles were supportive to the PAI, PTT, or fluorescence
capabilities of the dyes. In fact, the nanoparticle’s hydrophilic
exterior allowed the dyes to be used in aqueous solutions, and
thus, they were able to effectively deliver hydrophobic
heptamethine cyanine NIR dyes to cells and preserve their
PAI and PTT properties after cellular internalization.
After confirming sufficient cellular uptake, the ES2 cell
pellets, after 24 h incubation with 4h-NP and ICG (as a
positive control) at 10 μM, were subjected to PAI within the
2% agarose gel. The ultrasound images overlapped with PA
signals recorded for the 4h-NP- and ICG-treated cells were
detected above the negative controls of untreated cells as
unmixed (extracted) images (Figure 3C), represented by
specific colors (green for 4h-NP and red for ICG). These data
confirmed the cellular uptake shown by fluorescence imaging
(Figure 3B) as well as pronounced PA signal generation within
the cells by newly developed nanoparticles containing
heptamethine cyanine dye 4h-NP. The PA spectra (Figure
3D) generated from corresponding PA images (Figure 3C)
confirmed the presence of PA peaks for 4h-NP (green) and
ICG (red) in cells when compared to the PA peaks of the same
formulations in solutions. The 4h-NP PA peak at 820 nm was
maintained in the cellular environment, except for a slight
widening of the spectral peak. The ICG PA peak, on the other
hand, showed the pronounced broadness and a shift in the
spectra from 830 to 800 nm when measured in cells, as
compared to the one recorded in the solution. Thus, 4h-NP
demonstrated favorable PAI properties in vitro, as a sharper
and narrower PA peak promises definite identification by
spectral unmixing even at low molar concentrations.
EXPERIMENTAL SECTION
■
Materials. All chemicals and solvents were of American Chemical
Society grade or HPLC purity and were used as received. HPLC-
grade ethanol and tetrahydrofuran (THF) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). PEG-PCL (methoxy poly-
(ethylene glycol)-b-poly(ε-caprolactone), MW: 5−10k) was pur-
chased from Advanced Polymer Materials Inc. (Montreal, Canada).
All other chemicals were purchased from Fisher Scientific (Pittsburgh,
PA, USA) or Acros Organics (Pittsburgh, PA, USA). The reactions
were monitored using silica gel 60 F254 thin-layer chromatography
plates (Merck EMD Millipore, Darmstadt, Germany). The H, 13C,
1
and 19F NMR spectra were obtained using high-quality Kontes NMR
tubes (Kimble Chase, Vineland, NJ, USA) rated to 400 MHz and
were recorded on an Avance II spectrometer (Bruker, Billerica, MA;
400 MHz for 1H and 100 MHz for 13C) in DMSO-d6, acetone-d6, and
CDCl3. The high-resolution mass spectra were obtained at the
Georgia State University Mass Spectrometry Facility using a Q-TOF
micro (ESI-Q-TOF) mass spectrometer (Waters, Milford, MA, USA).
All compounds tested were >95% pure.
Synthesis of Dye-Loaded Nanoparticles. The nanoparticles
were produced with a solvent evaporation method.33,36,37 Briefly, 20
mg of poly(ethylene glycol)-block-poly(ε-caprolactone)methyl ether
(PEG-PCL) copolymer was solubilized in THF and introduced to the
NIR absorbing dye (0.6 mg) in a total volume of 2 mL of THF under
constant stirring conditions. Subsequently, water (2 mL) was added,
and THF was immediately removed using a Heidolph rotovap
(Schwabach, Germany). The obtained solution was filtered (0.2 μm
pore size membrane) to produce uniform nanoparticles. Nanoparticle
characterization, cellular cytotoxicity, and internalization were
assessed as reported previously by our lab (see the Supporting
Information).38,39
Assessment of Photoacoustic (PA) Capabilities. Photo-
acoustic properties of the dye-loaded nanoparticles were assessed in
water by injecting imaging nanoparticle-based agents (loaded with
4a−4ee) into a capillary tube (0.015 inches in interior diameter) and
sealing the ends. These were imaged via a Vevo LAZR system
(FUJIFILM VisualSonics, Toronto, Canada) using an LZ550
transducer to produce PA images. Within a defined region of interest
(ROI) on the image of interest, the 680−970 nm PA spectra were
generated at 5 nm increments. These spectral peaks were
subsequently normalized to 1.
CONCLUSIONS
■
Advances in biomedical imaging, like emerging PAI, continue
to be a major focus in developing platforms for early diagnosis
and therapeutic intervention treatment. Additionally, PTT
strategies have progressed as a stand-alone application or
theranostically in addressing disease states. As these modalities
move into the clinical field, optimization of novel contrast
agents with improved PAI and PTT properties will augment
their specificity and efficaciousness. Heptamethine cyanine
dyes are prime candidates in this field as they possess excellent
molecular features that permit them to be easily functionalized
and thus optimize the desired photophysical properties. In this
work, heptamethine cyanine dyes bearing cyclohexene in the
middle of the polymethine chain and open-chain dyes have
been synthesized, characterized, loaded into a polymeric
nanoparticle carrier, and analyzed for their photoacoustic and
photothermal capabilities. Structural modifications were
performed on the synthesized heterocycles to ensure different
In Vitro Photoacoustic Imaging. Human ovarian cancer cells, ES2
(ATCC, Manassas, VA), were seeded in T-25 flasks at a density of 0.7
× 106 cells/well and allowed to expand to ∼75% confluency. Cells
were then incubated with agents 4h-NP (10 μM) and ICG (10 μM).
Untreated ES2 cells were used as a control. After 24 h, cells were
8803
https://doi.org/10.1021/acs.jmedchem.1c00771
J. Med. Chem. 2021, 64, 8798−8805

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
trypsinized, washed thoroughly with DPBS, and pelleted. Next, cell
pellets were resuspended in the DPBS:2% activated agarose (1:1)
mixture (100 μL), and the resulting cell suspension was injected into
a recessed cavity within a cast 2% agarose gel for subsequent imaging.
Ultrasound and PA images were recorded on a Vevo LAZR
photoacoustic imaging system (LZ550 transducer, FUJIFILM Visual-
Sonics, Inc., Toronto, Canada) every 5 nm from the 680 to 970 nm
spectral range. Subsequently, regions of interest (ROIs) were around
imaged cell pellets and evaluated at the corresponding wavelength:
819 nm (for 4h-NP) and 830 nm (for ICG). PA signals obtained
from solution samples were used for unmixing (separating and
extracting the corresponding signal) to produce PA images of selected
colors, associated with the particular imaging agent. The nontreated
cell pellet within a cast 2% agarose gel was used as a control.
Diagnostics and Therapeutics, Georgia State University,
Atlanta, Georgia 30303, United States; orcid.org/0000-
0002-6475-0156; Email: mhenary1@gsu.edu
Authors
Anna St. Lorenz − Department of Pharmaceutical Sciences,
College of Pharmacy, Oregon State University, Portland,
Oregon 97201, United States
Emmanuel Ramsey Buabeng − Department of Chemistry,
Georgia State University, Atlanta, Georgia 30303, United
States; Center for Diagnostics and Therapeutics, Georgia
State University, Atlanta, Georgia 30303, United States
Oleh Taratula − Department of Pharmaceutical Sciences,
College of Pharmacy, Oregon State University, Portland,
Oregon 97201, United States; orcid.org/0000-0002-
6227-9110
Evaluation of the Photothermal Effect. For a temperature
increase evaluation, 300 μL of dye (4a−4ee)-loaded nanoparticles in
aqueous solutions (20 μM) in the 500 μL vials was exposed for 10
min to the NIR laser (780 or 808 nm, 0.6 W/cm2). The temperature
́
was recorded with a fiber optic thermal probe (Neoptix, Quebec,
Complete contact information is available at:
Canada). As a control, water was probed under the same irradiation
settings. The absorption of dye-loaded nanoparticle solutions was
evaluated prior to and after 10 min NIR laser treatment to evaluate
photostability.
In Vitro Photothermal Evaluation. ES2 cells were grown and
treated in T-75 flasks (3.2 × 106 cells/well) with 4h-NP (10 μM) and
ICG (10 μM) for 24 h. Then, cells were washed three times with
DPBS, trypsinized, and collected into the pellet. Next, the loose cell
pellets were irradiated with 808 nm laser (Wavespectrum Laser Group
Limited, China) light (0.6 W/cm2) for 10 min. Untreated cells were
used as controls. The temperature within the cell pellets was recorded
with a fiber optic thermal probe immediately after 10 min NIR laser
exposure.
https://pubs.acs.org/10.1021/acs.jmedchem.1c00771
Author Contributions
∥A.E.S. and E.R.B. contributed equally to this work.
Funding
This research was supported by the College of Pharmacy at
Oregon State University (OSU), the National Cancer Institute
of the National Institutes of Health under award numbers
R01CA237569 and R37CA234006, and the National Center
for Advancing Translational Sciences of NIH (KL2
TR002370).
General Procedure for Preparation of Salts 3a−3t. The
respective hydrazines (4.0 g, 22.25 mmol) were reacted with 3-
methylbutanone (3 mL, 28.04 mmol) in acetic acid and heated to 100
°C for 24 h. The resulting mixture was diluted with dichloromethane
followed by successive neutralization with sodium bicarbonate. The
combined extracts were concentrated under reduced pressure to
afford the substituted compounds 2. Subsequent alkylation of
compounds 2 in acetonitrile under reflux conditions for about 12−
18 h formed heterocyclic salts 3a−3t.4
General Procedure for the Synthesis of Heptamethine
Cyanine Dyes 4a−4ee. The various salts 3a−3t (2 molar equiv),
the linkers (1 molar equiv), and sodium acetate (2 molar equiv) were
dissolved in acetic anhydride and heated to 60 °C for 2−3 h. The
crude products were purified either via a column with eluting solvents
DCM:MeOH (10:1) or precipitated with diethyl ether, collected, and
washed with methanol to yield heptamethine dyes 4a−4ee as pure
samples.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
M.H. and E.B. would like to thank the Center for Diagnostics
and Therapeutics at Georgia State University for EB
fellowship. The authors thanks the Brains, Behavior Seed
Grant, The Atlanta Clinical and Translational Science Institute
Healthcare Innovation Seed Grant, and The Georgia Research
Alliance Ventures Phase 1 Grant for the support. M.H. and
E.B. thank Dr. Andrew Levitz for his synthetic procedures
reported in his dissertation and Mr. Matt Laramie for his initial
contribution to the project.
ABBREVIATIONS
■
ACN, acetonitrile; DLS, dynamic light scattering; EPR,
enhanced permeability and retention; HOAc, acetic aci; mV,
millivolts; NIR, near-infrared; PAI, photoacoustic imaging; PA,
photoacoustic; PDI, polydispersity index; PEG-PCL, poly-
(ethylene glycol)-b-poly(ε-caprolactone); PTT, photothermal
therapy; USI, ultrasound imaging; DCM, dichloromethane;
equiv., equivalent; THF, tetrahydrofuran; NMR, nuclear
magnetic resonance
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00771.
■
sı
1H NMR, 13C NMR, mass spectroscopy analysis, and
photophysical properties of all the tested dyes and dye-
loaded nanoparticles (PDF)
Molecular formula strings (CSV)
REFERENCES
■
AUTHOR INFORMATION
Corresponding Authors
(1) Gibbs, S. L. Near infrared fluorescence for image-guided surgery.
Quant. Imaging Med. Surg. 2012, 2, 177−187.
(2) Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y.
New strategies for fluorescent probe design in medical diagnostic
imaging. Chem. Rev. 2010, 110, 2620−2640.
■
Olena Taratula − Department of Pharmaceutical Sciences,
College of Pharmacy, Oregon State University, Portland,
Oregon 97201, United States; Email: olena.taratula@
oregonstate.edu
Maged Henary − Department of Chemistry, Georgia State
University, Atlanta, Georgia 30303, United States; Center for
(3) Troyan, S. L.; Kianzad, V.; Gibbs-Strauss, S. L.; et al. The
FLARE intraoperative near-infrared fluorescence imaging system: a
first-in-human clinical trial in breast cancer sentinel lymph node
mapping. Ann. Surg. Oncol. 2009, 16, 2943−2952.
8804
https://doi.org/10.1021/acs.jmedchem.1c00771
J. Med. Chem. 2021, 64, 8798−8805

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(4) Owens, E. A.; Hyun, H.; Tawney, J. G.; Choi, H. S.; Henary, M.
Correlating molecular character of NIR imaging agents with tissue-
specific uptake. J. Med. Chem. 2015, 58, 4348−4356.
(5) Stater, EP, Skubal, M, Tamura, R, Grimm, J. The present and
future of optical imaging technologies in the clinic: diagnosis and
therapy. In: Cheng, Z, editor. Fluorescent imaging in medicinal
chemistry. Cham: Springer International Publishing; 2020. p. 203−
223.
(6) Dost, T. L.; Gressel, M. T.; Henary, M. Synthesis and optical
properties of pentamethine cyanine dyes with carboxylic acid
moieties. Anal Chem Insights 2017, 12, 1177390117711938.
(7) Zhao, J.; Zhong, D.; Zhou, S. NIR-I-to-NIR-II fluorescent
nanomaterials for biomedical imaging and cancer therapy. J. Mater.
Chem. B, Mater. Biol. Med. 2018, 6, 349−365.
(8) Hatamimoslehabadi, M.; Bellinger, S.; La, J.; et al. Correlation of
photophysical properties with the photoacoustic emission for a
selection of established chromophores. J. Phys. Chem. C 2017, 121,
24168−24178.
(9) Guo, B.; Sheng, Z.; Hu, D.; et al. Molecular engineering of
conjugated polymers for biocompatible organic nanoparticles with
highly efficient photoacoustic and photothermal performance in
cancer theranostics. ACS Nano 2017, 11, 10124−10134.
(10) Hong, G.; Antaris, A. L.; Dai, H. Near-infrared fluorophores for
biomedical imaging. Nat. Biomed. Eng. 2017, 1, 0010.
(11) van der Vorst, J. R.; Schaafsma, B. E.; Hutteman, M.; et al.
Near-infrared fluorescence-guided resection of colorectal liver
metastases. Cancer 2013, 119, 3411−3418.
(12) Tummers, Q. R. J. G.; Verbeek, F. P. R.; Prevoo, H. A. J. M.;
et al. First experience on laparoscopic near-infrared fluorescence
imaging of hepatic uveal melanoma metastases using indocyanine
green. Surg. Innov. 2015, 22, 20−25.
(24) Zelepukin, I. V.; Yaremenko, A. V.; Yuryev, M. V.; et al. Fast
processes of nanoparticle blood clearance: Comprehensive study. J.
Controlled Release 2020, 326, 181−191.
(25) Illy, H.; Funderburk, L. Fischer indole synthesis. Direction of
cyclization of isopropylmethyl ketone phenylhydrazone. J. Org. Chem.
1968, 33, 4283−4285.
(26) Yuan, L.; Lin, W.; Song, J. Ratiometric fluorescent detection of
intracellular hydroxyl radicals based on a hybrid coumarin-cyanine
platform. Chem. Commun. 2010, 46, 7930−7932.
(27) Zimmermann, T.; Hennig, L. Ring transformations of
heterocyclic compounds.XXII . Pyrido[1,2- a ]indolium salts from
2-methyl-3 H -indoles by pyrylium mediated three carbon annelation.
J. Heterocycl. Chem. 2002, 39, 263−269.
(28) Capozza, M.; Blasi, F.; Valbusa, G.; et al. Photoacoustic imaging
of integrin-overexpressing tumors using a novel ICG-based contrast
agent in mice. Photoacoustics 2018, 11, 36−45.
(29) Egloff-Juras, C.; Bezdetnaya, L.; Dolivet, G.; Lassalle, H.-P. NIR
fluorescence-guided tumor surgery: new strategies for the use of
indocyanine green. Int. J. Nanomed. 2019, Volume 14, 7823−7838.
(30) Wang, H.; Li, X.; Tse, B. W.-C.; et al. Indocyanine green-
incorporating nanoparticles for cancer theranostics. Theranostics 2018,
8, 1227−1242.
(31) Kanazaki, K.; Sano, K.; Makino, A.; et al. Feasibility of
poly(ethylene glycol) derivatives as diagnostic drug carriers for tumor
imaging. J. Controlled Release 2016, 226, 115−123.
(32) Sun, X.; Wang, G.; Zhang, H.; et al. The blood clearance
kinetics and pathway of polymeric micelles in cancer drug delivery.
ACS Nano 2018, 12, 6179−6192.
(33) Duong, T.; Li, X.; Yang, B.; et al. Phototheranostic
nanoplatform based on a single cyanine dye for image-guided
combinatorial phototherapy. Nanomedicine 2017, 13, 955−963.
(34) Nomura, S.; Morimoto, Y.; Tsujimoto, H.; et al. Highly reliable,
targeted photothermal cancer therapy combined with thermal
dosimetry using a near-infrared absorbent. Sci. Rep. 2020, 10, 9765.
(35) Doughty, A.; Hoover, A.; Layton, E.; Murray, C.; Howard, E.;
Chen, W. Nanomaterial applications in photothermal therapy for
cancer. Materials (Basel) 2019, 12, 779.
(36) Marsden, H. R.; Gabrielli, L.; Kros, A. Rapid preparation of
polymersomes by a water addition/solvent evaporation method.
Polym. Chem. 2010, 1, 1512.
(37) Han, X.; Taratula, O.; Taratula, O.; et al. Biodegradable
hypericin-containing nanoparticles for necrosis targeting and fluo-
rescence imaging. Mol. Pharmaceutics 2020, 17, 1538−1545.
(38) Li, X.; Schumann, C.; Albarqi, H. A.; et al. A Tumor-activatable
theranostic nanomedicine platform for NIR fluorescence-guided
surgery and combinatorial phototherapy. Theranostics 2018, 8, 767−
784.
(39) Schumann, C.; Chan, S.; Khalimonchuk, O.; et al. Mechanistic
nanotherapeutic approach based on siRNA-Mediated DJ-1 protein
suppression for platinum-resistant ovarian cancer. Mol. Pharmaceutics
2016, 13, 2070−2083.
(13) Ashitate, Y.; Vooght, C. S.; Hutteman, M.; Oketokoun, R.;
Choi, H. S.; Frangioni, J. V. Simultaneous assessment of luminal
integrity and vascular perfusion of the gastrointestinal tract using dual-
channel near-infrared fluorescence. Mol. Imaging 2012, 11,
7290.2011.00048.
(14) Ashitate, Y.; Levitz, A.; Park, M. H.; et al. Endocrine-specific
NIR fluorophores for adrenal gland targeting. Chem. Commun. 2016,
52, 10305−10308.
(15) Hyun, H.; Owens, E. A.; Wada, H.; et al. Cartilage-specific
near-infrared fluorophores for biomedical imaging. Angew. Chem. Int.
Ed. Engl. 2015, 54, 8648−8652.
(16) Hyun, H.; Park, M. H.; Owens, E. A.; et al. Structure-inherent
targeting of near-infrared fluorophores for parathyroid and thyroid
gland imaging. Nat. Med. 2015, 21, 192−197.
(17) Hyun, H.; Wada, H.; Bao, K.; et al. Phosphonated near-infrared
fluorophores for biomedical imaging of bone. Angew. Chem. Int. Ed.
Engl. 2014, 53, 10668−10672.
(18) Kim, S. H.; Park, G.; Hyun, H.; et al. Near-infrared lipophilic
fluorophores for tracing tissue growth. Biomed. Mater. 2013, 8,
No. 014110.
(19) Owens, E. A.; Henary, M.; El Fakhri, G.; Choi, H. S. Tissue-
specific near-infrared fluorescence imaging. Acc. Chem. Res. 2016, 49,
1731−1740.
(20) Bashmakova, N. V.; Shaydyuk, Y. O.; Przhonska, O. V.; et al.
Spectroscopic study and quantum chemical analysis of the asym-
metrical cationic polymethine dye versus its symmetrical analogues.
Mol. Cryst. Liq. Cryst. 2014, 590, 199−212.
(21) Mishra, A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera,
G. B. Cyanines during the 1990s: a review. Chem. Rev. 2000, 100,
1973−2012.
(22) Pisoni, D. S.; Todeschini, L.; Borges, A. C. A.; Petzhold, C. L.;
Rodembusch, F. S.; Campo, L. F. Symmetrical and asymmetrical
cyanine dyes. Synthesis, spectral properties, and BSA association
study. J. Org. Chem. 2014, 79, 5511−5520.
(23) El-Shishtawy, R. M.; Asiri, A. M.; Basaif, S. A.; Rashad, S. T.
Synthesis of a new beta-naphthothiazole monomethine cyanine dye
for the detection of DNA in aqueous solution. Spectrochim. Acta. A
Mol. Biomol. Spectrosc. 2010, 75, 1605−1609.
8805
https://doi.org/10.1021/acs.jmedchem.1c00771
J. Med. Chem. 2021, 64, 8798−8805