Article

Cite This: J. Phys. Chem. C 2017, 121, 22958-22968 pubs.acs.org/JPCC

Specific Interaction of Tricyclic Antidepressants with Gold and Silver

Nanostructures as Revealed by Combined One- and Two-Photon
Vibrational Spectroscopy
Vesna Ž ivanović,†,‡ Fani Madzharova,† Zsuzsanna Heiner,†,‡ Christoph Arenz,†,‡ and Janina Kneipp*,†,‡
†
Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany
‡
School of Analytical Sciences Adlershof SALSA, Humboldt-Universität zu Berlin, Albert-Einstein-Str. 5−11, 12489 Berlin, Germany
*
S Supporting Information
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ABSTRACT: We report two-photon excited nonresonant surface-enhanced hyper

Raman scattering (SEHRS) spectra of tricyclic antidepressant (TCA) molecules during
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their interaction with biocompatible gold nanostructures and with silver nanostruc-
tures. The SEHRS spectra of amitriptyline, desipramine, and imipramine are compared
with surface-enhanced Raman scattering (SERS) spectra on both kinds of
nanoparticles, obtained with excitation at 532 and 785 nm. The SEHRS spectra of
the TCA molecules show several intense contributions by infrared-active vibrations.
Combining SEHRS with SERS therefore enables a comprehensive vibrational
characterization of the interaction of the molecules with the nanostructures. SEHRS
and SERS data indicate that the molecules interact with the silver nanostructures
mainly via their ring moiety. In contrast, in the interaction with gold, the
methylaminopropyl side chain plays a very important role, along with parts of the
ring system. It is possible to obtain the spectra of the molecules with near-infrared excitation and with gold nanoparticles in cell
culture media. The spectral signatures of the drug molecules collected at low pH values characteristic of late endosomal stages or
of acidified tissues are very stable and show only small changes in the interaction of the TCA with the gold nanoparticles. The
results will help to develop tools for the characterization of new nanoparticle-based drug delivery platforms in real biological
environments.

■ INTRODUCTION
Tricyclic antidepressants (TCAs) are an important group of
drugs. Initially used for the clinical treatment of major
depression disorders, TCA have been suggested as potential
treatments for a variety of other diseases, such as neuropathic
pain disorders1 or irritable bowel syndrome.2 Furthermore,
TCA molecules have anti-inflammatory, antimicrobial,3 and
antitumoral activity.4 They were shown to have an influence on
lipid metabolism,5 specifically, TCAs reduce the acitivity of the
acid sphingomyelinase and acid ceramidase enzymes in the
lysosomes.6,7 A destabilization of the lysosomal membranes has
been proposed as a main cause of cell death and ultimately
antitumoral activity of the compounds,8−11 yet details about the
molecular interaction of the TCA molecules with the biological
environment and different bioorganic molecules must be
elucidated.
Vibrational spectra, particularly Raman scattering and
infrared (IR) absorption spectra, can provide detailed
information about the structure and interactions of active
substances and biological molecules12−14 and have become
important tools for studying complex biological samples, such
as cells and tissues in healthy and diseased state.15,16 The two-
photon excited analogue of the spontaneous Raman scattering
process, hyper-Raman scattering (HRS), can be used to obtain
complementary vibrational information by probing infrared-
active and silent vibrational modes that cannot be observed in
the normal, one-photon excited Raman spectrum.17−19 The
intrinsically weak HRS process becomes very strong when the
local optical fields of plasmonic nanostructures are employed in
surface-enhanced hyper Raman scattering (SEHRS).20−24 As
we and others have shown in the last years, by SEHRS,
vibrational information complementary to surface-enhanced
Raman scattering (SERS) spectra can be obtained from organic
molecules and complex biosamples,25−31 and specifically from
their interaction with the surface of metal nanostructures.
Recent findings suggest that SEHRS is much more sensitive
toward small changes in molecular structure and local adsorbate
environments than SERS is.28,32,33 As will be shown here as
well, SEHRS can provide additional chemical information.
Because some of the vibrations and molecular groups are
observed in only one spectrum and not the other, the two kinds
of vibrational spectra provide more comprehensive information
about the TCA molecules and their orientation with respect to
the gold or silver surface than SERS spectra alone. Combining
SEHRS and SERS can give better insight into the interaction of
Received: August 11, 2017
Revised: September 15, 2017
Published: September 18, 2017

© 2017 American Chemical Society 22958 DOI: 10.1021/acs.jpcc.7b08026

J. Phys. Chem. C 2017, 121, 22958−22968
The Journal of Physical Chemistry C
the active molecules with the nanoparticles as well as with other
bioorganic compounds, in vitro and in cells.
In this paper, we report SEHRS spectra of the three tricyclic
antidepressant (TCA) molecules amitriptyline (Ami), desipr-
amine (Des), and imipramine (Imi) (Figure 1) and discuss
Figure 1. Structure and labeling of antidepressant molecules,
imipramine (Imi), desipramine (Des), and amitriptyline (Ami).
them in comparison to their one-photon excited SERS spectra.
The SERS spectra of the molecules have been published
previously,14,30,34 and propositions regarding their interaction
with the surface of silver nanoparticles have been made.30 Using
the two-photon excited SEHRS spectra here, and by employing
both gold and silver nanoparticles, the comprehensive vibra-
tional characterization reveals that the interaction of the
molecules with nanostructure surfaces, as well as with other
molecular species, strongly differs for silver and gold surfaces
and is very characteristic even at varied concentration and pH.
With potential applications in cells and tissues in mind, we
report spectra from biocompatible gold nanostructures,35
obtained under conditions that are relevant for experiments
in biological environments, such as cell culture media. Our
findings may have implications for the application of combined
one- and two-photon vibrational characterization in therapeutic
drug monitoring, studies of drug-biomolecule interactions, as
well as for the construction of new multifunctional nanocarriers
based on gold nanoparticles.
■ MATERIALS AND METHODS
Desipramine hydrochloride, imipramine hydrochloride, ami-
triptyline hydrochloride, gold(III) chloride hydrate, silver
nitrate, sodium hydroxide, sodium chloride, and hydrochloric
acid were purchased from Sigma-Aldrich. Fetal calf serum
(FCS) and Dulbecco’s Modified Eagle Medium (DMEM) were
purchased from Biochrom. All solutions were prepared using
Milli-Q water.
Different batches of gold nanoparticles with an average size
of 30 and 60 nm termed here Au30 and Au60, respectively,
were obtained by the reduction of gold(III) chloride hydrate
with sodium citrate according to the protocols reported in refs
36 and 37 yielding a concentration for Au30 of 4 × 10−10 M
and for Au60 of 7 × 10−11 M. Silver nanoparticles (AgNP) were
prepared according to ref 36 by reduction of silver nitrate with
sodium citrate, resulting in a nanoparticle concentration of
2 × 10−11 M.
Desipramine hydrochloride, imipramine hydrochloride, and
amitriptyline hydrochloride were separately dissolved in water
and mixed with the nanoparticles/nanoaggregates at a volume
ratio of 1:10, yielding final concentrations in the range from
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Article
10−4 M to 10−7 M, depending on the particular experiment.
Concentrations are given with the respective spectra in Results
and Discussion. For experiments in the cell culture media, the
nanoparticles with the molecules were mixed with Dulbecco’s
Modified Eagle Medium supplemented with 10% of fetal calf
serum (FCS) in a final concentration of TCA molecules of
9 × 10−7 M. To adjust pH values, NaOH was used.
The Raman setup was described in ref 32 before. Surface-
enhanced hyper-Raman (SEHRS) spectra were excited at
1064 nm with an excitation intensity of 2.1 × 1011 W cm−2
using a mode-locked laser producing 7 ps pulses with a
repetition rate of 76 MHz. One-photon excitation at 532 nm
was generated as second harmonic signal from the picosecond
1064 nm laser; the applied intensity was 1.2 × 1010 W cm−2.
Excitation at 785 nm was provided by a CW laser with an
excitation intensity of 2.0 × 105 W cm−2. The sample solution
was placed in a microcontainer, and the excitation light was
focused on the samples through a microscope objective.
SEHRS spectra were accumulated for 5 min, while SERS
spectra were accumulated for durations of 1−5 s. The Raman
light was collected in backscattering geometry and detected by
a liquid nitrogen-cooled CCD (Horiba, Munich, Germany).
After frequency calibration using a spectrum of toluene, the
SEHRS spectra were background corrected using the method
provided in ref 38. Most spectral analyses were carried out on
averaged spectra; averages were separately calculated from 100
SERS spectra and 30 SEHRS spectra.
■ RESULTS AND DISCUSSION
In order to investigate the interactions of the antidepressant
molecules with gold and silver nanoparticles, both one- and
two-photon excited surface-enhanced Raman scattering, that is,
SERS and SEHRS, were employed. Both, gold and silver
nanostructures were synthesized using citrate as reducing agent
and stabilizer. Transmission electron micrographs of the
nanostructures used in the experiments are shown in Figure
2, together with ultraviolet−visible (UV−vis) absorbance
spectra at the condition of the respective experiment, that is,
Figure 2. Transmission electron micrographs of (A) silver nano-
particles (Ag), (B) the gold nanoparticles with average size 30 nm
(Au30), and (C) gold nanoparticles with average size 60 nm (Au60).
(D) UV−vis absorbance spectra of the AgNP (black line), Au30 (red
line), and Au60 (blue line) with addition of desipramine (Des).
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Figure 3. (A) SEHRS and (B) SERS spectra of the antidepressants imipramine, desipramine and amitriptyline obtained with silver and gold
nanostructures. Concentration of the TCA moelcules: 9 × 10−5 M for Au and 9 × 10−4 M for Ag nanostructures. (A) Excitation wavelength,
1064 nm; intensity, 2.1 × 1011 W cm−2; acquisition time, 5 min. (B) Excitation wavelength, 532 nm; laser intensity, 1.2 × 1010 W cm−2; acquisition
time, 5 s. Scale bars: (A) SEHRS, 0.1 cps; (B) SERS, 50 cps.

in the presence of the TCA molecules. The silver nanoparticles
have a size of 110 nm (Figure 2A). Two different samples of
citrate-stabilized gold nanoparticles were used, one with a mean
diameter of 30 nm (Figure 2B), the other with a diameter of
60 nm (Figure 2C). Figure 2D shows the absorbance of the
plasmonic nanostructures in the presence of one of the TCA
molecules, desipramine. Prior to the addition of the TCA
molecules, the citrate-reduced silver and gold nanoparticles
exhibit the typical absorbance maxima around 420 nm for silver,
and 530 and 542 nm for the two different batches of gold
nanoparticles, respectively (Figure S1 in the Supporting
Information). After the addition of the TCA molecules, all
nanoparticles indicate absorbance by a broad, extended
plasmon resonance (Figure 2D). A change in color is also
observed by eye when the TCA molecules are added.
As shown in Figure 3A, it was possible to obtain high-quality
nonresonant SEHRS spectra of all three compounds, using
both silver and gold nanoparticles. The SEHRS spectra of
amitriptyline (Ami), desipramine (Des), and imipramine (Imi)
(Figure 1) were excited at a wavelength of 1064 nm (Figure
3A), and corresponding one-photon excited SERS spectra using
532 nm excitation were measured (Figure 3B). The SEHRS
22960
signals with silver and gold nanoparticles are on the same order
of magnitude; this is in accordance with the main contribution
of the SEHRS enhancement being discussed to result from the
local fields of nanoaggregate structures,39,40 whose formation is
indicated in the absorbance spectra (Figure 2D). To date,
relatively few reports on SEHRS spectra using gold nano-
particles are known.41,42 In the case of the system studied here,
SEHRS and SERS spectra obtained with gold nanostructures
are of particular interest because of the biocompatibility of the
gold and its potential use as drug carrier.
SEHRS and SERS Spectra Give Complementary
Information. Both one- and two-photon excited spectra with
the two types of nanoparticles are characteristic of the three
TCA molecules, and the SERS spectra on silver nanoparticles
obtained here (Figure 3B, second, fourth, and bottom traces)
are in good agreement with those reported previously.30,34 We
obtained very similar SEHRS spectra (Figure 3A) and very
similar SERS spectra (Figure 3B) of the three different drugs,
and as discussed in detail below, the spectra indicate an
interaction characteristic of gold and silver nanostructures (cf.
every second spectrum in Figure 3A,B). At first glance the
spectral differences between spectra on silver and gold appear
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greater than the differences between the three molecules. Table 2. Raman Shift Values in the SEHRS and SERS
Nevertheless, as will be shown, the spectra of the molecules Spectra of Desipramine and Assignment to Vibrations of the
display significant qualitative differences, depending on their Desipramine Molecule, Based on Refs 30, 43, 67, and 68
structure and specific interaction with a particular kind of
Raman shift
nanoparticle (cf. every second spectrum in Figure 3A,B). Band (cm−1)
assignments for the spectra of Ami, Des, and Imi are provided
SEHRS SERS assignmentsa
in Tables 1, 2, and 3, respectively. After common spectral
1630 − str (CC/RII)
Table 1. Raman Shift Values in the SEHRS and SERS − 1597 i-p bend (CC/RI,RII)
Spectra of Amitriptyline and Assignment to Vibrations of the 1575 1576 str (CC/RI), str (CC/RII)
Amitriptyline Molecule, Based on Refs 30, 43, 67, and 68 1487 − bend (CH2)
1475 1472 def (CH2)3, def (CH3)
Raman shift − 1440 sciss (CH2/aliph)
(cm−1)
1386 1363 symm def (CH3),
SEHRS SERS assignmentsa 1329 1335 i-p bend (CH2/aliph)
1623 1614 i-p bend (CC/RI,RII) 1296 1295 str (CC/RIII, bridge bond)
1590 1590 str (CC/RI,II), def (CH/RI,II) 1280 − i-p bend (CH2)
1575 1556 str (CC/RI,II) 1232 1229 str (NC/RIII), rock (CH/RI), twist (CH2/RIII)
1487 1486 bend (CH2) 1206 1207 str (CC/RIII), i-p bend (RI,II), str (CC/RI,II)
1464 1462 bend (CH2/aliph), str (RI) 1155 1152 rock (CH/RI,II)
− 1438 bend (CH2/aliph), str (RI) 1107 − bend (CH/RI,II)
− 1428 i-p bend (NC/Me) 1055 1056 str (CC/RI), rock (CH/RI)
− 1378 i-p bend (CH2) 1039 1040 str (CC/aliph), str (CC/RII)
1360 1363 rock (CH2/aliph), str (CC/RIII), def CH3 1018 − str (CC/RI,II)
− 1335 bend (CH2/aliph) − 973 str (CC/RIII), rock (CH2/RIII)
1295 1290 str (CC/RIII, bridge bond) − 865 o-o-p bend (CH/RI)
1280 1273 i-p bend (CH/RI,II), bend (CH2/aliph), str (RI) − 846 bend (CH2)
− 1221 i-p bend (RI,II) − 812 o-o-p bend (CH/RI,II)
1206 1207 str (CC/RIII), i-p bend (RI,II), rock (CH/RII), str (CC/ − 791 bend (CH2), bend (RI)
RI,II) − 774 twist (RI,II), wag (N2H), i-p bend (RI,II), str (CC/RIII)
1177 1170 i-p bend (CH2) 765 − def CH (RI,II)
1161 − rock (CH/RI,II) − 685 i-p bend (RI,II), str (CC/RIII), twist (RI,II)
− 1159 str (CC/RIII), rock (CH/RI), i-p bend (RI,II), str (CC/ 677 673 i-p bend (RI,II)
RI,II)
622 620 twist (RI,II), i-p bend (RIII)
− 1055 str (CC/RI), rock (CH/RI)
585 585 twist (RI,II,III)
1039 1040 str(CC/aliph), rock (CH/RII)
566 562 i-p bend (RI,II,III), wag (N1C/aliph)
1018 − str (CC/RI,II)
− 538 i-p bend (CH2), twist (RI)
− 973 str (CC/RIII), rock (CH2/RIII), i-p bend (RI)
− 496 twist (RI,II,III), i-p bend (RI,II), wag (N1C/aliph)
− 870 o-o-p bend (CH/RI,II) a
782 791 twist (RI,II), wag (N2H), i-p bend (RI,II), str (CC/RIII) str, stretching; def, deformation; bend, bending; sciss, scissoring; wag,
wagging; rock, rocking; twist, twisting; i-p bend, in-plane bending;
− 846 i-p bend (CH2)
o-o-p bend, out-of-plane bending.
− 812 o-o-p bend (CH/RI,II)
775 774 twist (RI,II), wag (N2H), i-p bend (RI,II), str (CC/RIII)
724 710 twist (RI,RII) two-photon excited case, although with varied relative
− 685 i-p bend (RI,II), str (CC/RIII) intensities. This similarity of the SEHRS and the SERS spectra
633 635 def (RI,II) is expected, because the interaction of the molecules with the
− 620 twist (RI,II), i-p bend (RIII) nanoparticles’ surface lowers their symmetry further, specifically
585 585 twist (RI,II,III) of the ring moieties. As examples, contributions by the C−C
− 562 i-p bend (RI,II,III)1 stretching modes, together with C−H deformation vibrations
533 533 i-p bend (RII), twist (RI,II), str (CC/RIII), i-p bend (CC/ of the rings, are found in most examples, as indicated by the
RIII)
bands around 1206 cm−1, e.g. in the spectra obtained with the
− 485 in-plane bend (RI,II)
silver nanoparticles (second, fourth, and bottom traces in
− 445 twist (RI,II,III)
a
Figures 3A and 3B), and at ∼1160 cm−1 (all spectra in Figure
str, stretching; def, deformation; bend, bending; sciss, scissoring; wag, 3A,B). Similarly, a C−C stretching vibration of the seven-
wagging; rock, rocking; twist, twisting; i-p bend, in-plane bending; membered ring at ∼1295 cm−1 is found both in SEHRS and
o-o-p bend, out-of-plane bending.
SERS spectra, when the molecules interact with a silver surface.
Furthermore, C−C stretching from the aliphatic tail at
features for all three molecules are evaluated first, some further ∼1040 cm−1 appears in both SERS and SEHRS spectra, with
specifics in the SEHRS spectrum of each TCA are discussed in very similar relative intensity, except in SEHRS of Des and Imi
a separate paragraph. on gold nanostructures.
The respective SEHRS and SERS spectra differ greatly for Even though the SEHRS spectra share many of the bands
each molecule and type of nanostructure (compare each line in with the SERS spectra, they are very different with respect to
the combined Figure 3A,B) over the whole spectral range. many of them as well. A large number of modes in the
Many of the bands are present in both the one-photon and the fingerprint region of the spectra occur either in the SEHRS or
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Table 3. Raman Shift Values in the SEHRS and SERS dibenzazepine ring at 973 cm−1,30 probably of a six-membered
Spectra of Imipramine and Assignment to Vibrations of the ring, this band is not found in any of the SEHRS spectra. This
Imipramine Molecule, Based on Refs 30, 43, 67, and 68 observation is in accord with the absence of bands of this and
similar modes in the SEHRS spectra of other organic molecules
Raman shift
(cm−1) in our previous studies.27,28
Vice versa, a strong band at 765 cm−1 is particularly
SEHRS SERS assignmentsa
enhanced in SEHRS of Des and Imi, but is absent from their
− 1590 i-p bend (CC/RI,RII) SERS spectra (Figure 3A,B, red and black traces). It is not seen
1575 1576 str (RI,II) in the normal Raman spectra either (Figure S2), but it makes a
− 1556 str (RI,II) very intense contribution to the IR spectrum of Des. There, the
1487 − bend (CH2) band is assigned to a deformation vibration of the −CH groups
1475 1472 def (CH2)3, def (CH3) in the aromatic rings. Furthermore, the SEHRS spectra,
− 1440 i-p bend (Me), sciss (CH2/aliph) specifically on silver nanoparticles, show a strong signal at
1386 1363 symm def (CH3) 585 cm−1, which is neither visible in the SERS data of Figure 3B
1333 1329 i-p bend (CH2/aliph) nor in the normal Raman spectra (Figure S2). The IR
1296 1295 str(CC/RIII, bridge bond) spectrum, however, clearly reveals a band at 585 cm−1. It is
1284 − i-p bend(CH2)
possible that an in-plane bending vibration of dibenzazepine
1232 1229 str (NC/RIII), rock (CH2/aliph), twist (CH2/aliph), str
(NC/aliph) ring is probed by SEHRS here, which would be specifically
− 1221 i-p bend (RI,II) enhanced upon an interaction of the ring moieties with the
1206 1202 str (CC/RIII), i-p bend (RI,II) silver surfaces that was predicted in ref 30. Furthermore, the
1177 1167 i-p bend (CH2) SEHRS spectra are all characterized by an intense band at
1155 1156 rock (CH/RI,II) 1487 cm−1, which we assign to a deformation mode of CH2
1055 − str (CC/RI), str (NC/aliph) group(s) of the aliphatic chain, observed at this frequency also
1039 1040 str (CC/aliph), sciss (CH2/aliph), i-p bend(NC/Me) in IR spectra43 (Figure 3A, first, third, and fifth traces). In the
− 1030 str(CC/aliph), sciss(CH2/aliph), i-p bend(NC/Me) SERS spectra in this frequency range, other deformation modes
− 973 str (CC/RIII), rock (CH2/RIII) of CH2 groups at lower frequency, from both the seven-
− 861 o-o-p bend (CH/RI), i-p bend (CH2) membered ring and the aliphatic chain, dominate this spectral
− 846 i-p bend (CH2) region, with contributions around ∼1462 and ∼1440 cm−1
− 812 o-o-p bend(CH/RI,II) (Figure 3B, first, third, and fifth traces).30,43 Also in the higher-
− 787 o-o-p bend(CH/RI), bend(CH2) frequency range, with a different scaling shown in the Figure
765 − twist (RI,II), wag (N2H), i-p bend (RI,II), str (CC/RIII) S3, C−C stretching modes of the dibenzazepine ring, which
− 685 i-p bend (RI,II), str (CC/RIII), twist (RI,II) have very low intensity in the normal Raman spectra (Figure
677 673 i-p bend (RI,II) S2) but are also strong in the IR spectra, e.g., the mode at
622 620 twist (RI,II), i-p bend (RIII) 1575 cm−1, become very pronounced. The high similarity of the
585 585 twist (RI,II,III) SEHRS spectra with IR data is in accord with previous
566 562 i-p bend (RI,II,III), wag (N1C) observations in SEHRS experiments with other organic
− 538 i-p bend (CH2), twist (RI) molecules27,28,33,44−46 and with predictions according to the
− 496 i-p bend (CH2/aliph), rock (NC) selection rules for the hyper Raman process that can probe IR
a
str, stretching; def, deformation; bend, bending; sciss, scissoring; wag, active or even silent vibrational modes.
wagging; rock, rocking; twist, twisting; i-p bend, in-plane bending; SEHRS Spectrum of Amitriptyline. Figure 3A shows the
o-o-p bend, out-of-plane bending. SEHRS spectra of amitriptyline (Ami) obtained with gold and
silver nanostructures separately (blue traces in Figure 3A). For
in the SERS spectrum, or have very different relative intensities comparison, the one-photon excited SERS spectra of the
in both spectra, illustrating the complementarity of the spectral identical samples are shown in Figure 3B (blue traces). The
information. The fact that some vibrations contribute to the assignments of the bands are given in Table 1. As stated above,
SEHRS spectrum of a molecule when interacting with, e.g., gold the spectra show intense contributions from vibrations of the
nanoparticles, but to the SERS spectrum of the same molecule dibenzazepine ring of the molecule, and also from the aliphatic
only with silver nanoparticles, evidences the very sensitive chain. The dominating band at 1487 cm−1 in the SEHRS
probing of local surface interaction by both methods and spectra of the two CH2 groups of the aliphatic chain is
indicates the strong influence that the interaction with the complemented by smaller contributions that originate from
nanoparticles exerts on molecular symmetry. other vibrations of the chain. Furthermore, an intense band at
A very obvious difference between SERS and SEHRS spectra 1206 cm−1 in the spectrum obtained on the gold nanoparticles,
common to all three TCA molecules is the absence of the assigned to the C−C stretching vibration in the seven-
intense signal of ring deformation mode at 685 cm−1 in the membered ring, is observed (Figure 3A, first spectrum). The
SEHRS spectra on silver nanoparticles. Similarly, on gold relative intensity of this C−C stretching vibration with respect
nanoparticles, the very intense bands at 846 and 812 cm−1 to other C−C stretching is similar to that of the mode at
(Figure 3B) that we assign to N−C deformation vibrations of 1623 cm−1 (compare Figure S3A, first trace to the other traces)
the methylaminopropyl side chain are absent from the SEHRS and for example also is higher on gold nanoparticles than on
spectra. In the normal Raman spectra, these bands, specifically silver. Another SEHRS signal that we assign to the
the former two, are also very pronounced (Figure S2). In dibenzazepine ring is found at 782 cm−1; it has a higher
similarity to the SEHRS spectra, the bands are absent in IR frequency than the CH deformation of the phenyl rings in Imi
spectra, as was reported for the case of Des.43 While most of and Des discussed above and a stronger contribution on silver
the SERS spectra display a band of a breathing vibration of the nanoparticles. Its position suggests a contribution from a C−C
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stretching mode of the seven-membered ring, which in Ami is
lacking a nitrogen atom, hence providing an additional C−C
bond compared to Des and Imi. In addition to the bands at 560
and 585 cm−1, another in-plane mode of the phenyl rings and
the seven-membered ring is present at 533 cm−1 in the Ami
SEHRS spectrum on silver nanoparticles, confirming the
previous observation that the interaction of the ring system
with silver nanoparticles differs from that of Des and Imi.30
SEHRS Spectra of Desipramine and Imipramine. The
SEHRS spectra of desipramine (Des, red traces) and
imipramine (Imi, black traces) obtained with gold and silver
nanostructures are shown in Figure 3A. Figure 3B contains the
corresponding SERS spectra excited at 532 nm. Compared to
the spectra of Ami, the SEHRS spectra of Des and Imi share
many characteristics, specifically regarding their interaction with
the silver and the gold nanoparticles. The band assignments of
the SEHRS and SERS spectra of Des and Imi are listed in
Tables 2 and 3, respectively.
The presence of a nitrogen atom in the seven-membered ring
of both molecules leads to several vibrational modes of the
N−C bonds, specifically the stretching vibration at 1232 cm−1,
which is quite pronounced in all SEHRS spectra but in the
SERS spectra clearly visible only when Des and Imi interact
with silver nanostructures. Similarly, the C−C bridge bond
vibration of the ring at 1295 cm−1 is weak only in the SERS
spectra on silver nanostructures, but very strong in all the
SEHRS spectra (compare Figure 3A, last four spectra with
Figure 3B, fourth and sixth traces). As a very specific feature of
the SEHRS spectra, the −CH deformation vibration of the
phenyl rings at 765 cm−1 gives strong signals. While the
breathing vibrational mode at 973 cm−1 is absent from the
SEHRS spectra, different in-plane bending modes of the phenyl
rings at 1206 and at 622 cm−1 are very pronounced in the
SEHRS spectra.
Also, the signals in the methylaminopropyl side chains of the
molecules differ from those known for Ami. In addition to the
intense signal at 1487 cm−1 of CH2 groups, vibrational modes
that are assigned to the methyl groups are visible, for example,
the CH3 symmetric deformation vibration at 1386 cm−1 and a
contribution at 1475 cm−1, assigned to deformation vibrations
of both CH2 and CH3 groups. Both bands have higher intensity
in the SEHRS spectra obtained with gold nanostructures. In
contrast, in the SERS spectra, the CH3/CH2 deformation at
1363 cm−1 is relatively weak and found only on the gold
nanoparticles, pointing toward an interaction of the methyl-
aminopropyl side chain with the gold surface. An important
contribution to the band at 1363 cm−1 coming from the methyl
groups is inferred from the presence of the band also in the
SERS spectra of Ami (blue traces in Figure 3B), where much
less signal from CH2 can be expected, but where two CH3
groups are terminating the chain. Furthermore, a band at
1325 cm−1 in Des and at 1333 cm−1 in Imi, assigned to a
bending vibration of the CH2 groups in the aliphatic chain, is
prominent in the spectra obtained with gold nanoparticles
(Figure 3A, third and fifth spectra). In the SERS spectra
measured under these conditions, a signal at 1335 cm−1 that we
assign to this vibration is found as well (Figure 3B, third and
fifth spectra). The different contributions of the C−H
deformation modes in the alkyl chains in the SEHRS and
SERS spectra indicate very clearly the presence of different
kinds of specific interactions of the CH2 groups with the
nanoparticle surfaces.
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The SEHRS spectra show several smaller differences between
Imi and Des, for example, the band at 1177 cm−1 in the Imi
spectrum and the band at 1155 cm−1 in the Des spectrum. In
both molecules, we assign it to the rocking of the CH from the
phenyl rings,30 and this difference being pronounced on the
silver nanoparticles underpins the conclusion of a strong
interaction of the ring system with the silver surface. Also, C−C
stretching modes of the phenyl ring around 1107 cm−1 and at
1055 cm−1 display slightly different contributions. On the gold
nanoparticles, only very weak or no bands at this position are
present.
Interaction of the Molecules with Gold and Silver
Nanostructures. Considering the high sensitivity of the
SEHRS and the SERS spectra with respect to molecular
interaction and surface environmental changes, the great
differences that become evident during the discussion of the
SEHRS and SERS spectra obtained on gold and silver
nanoparticles must be the result of a varied interaction of the
molecules with the surfaces of both kinds of nanostructures. In
the SERS data, the appearance and relative intensity of a certain
band can provide information on the orientation of the
molecules.47−49 Specifically, the vibrational modes of the
aromatic rings give this information based on the relative
intensity of in-plane and out-of-plane vibrations.49,50 In SEHRS,
relying on other selection rules, changes in adsorbate
orientation can lead to much more pronounced changes in
the spectra than in SERS,22,51,52 and the possibility of probing
other adsorbate species and/or interaction sites on the same
surface28,32 can add a wealth of information. Because of the
complementarity of the SEHRS and SERS spectra, we use both
together to discuss the differences in the interaction of the TCA
molecules. Apart from the excitation of SERS spectra at
532 nm, (Figure 3B), Figure S4 also shows SERS spectra that
were obtained with an excitation wavelength of 785 nm on both
metal nanostructures.
On the silver surface, the SEHRS spectra of all molecules
contain pronounced signals assigned to several vibrational
modes of the ring systems, specifically the in-plane bending
modes of the phenyl rings at 622, 585, and 566 cm−1 (see
Figure 3B, second, fourth, and sixth traces). Their higher
intensity on the silver nanostructures suggests an interaction of
the rings with the surface. On the silver nanostructures, the
strong band at 765 cm−1 of the −CH deformation of the phenyl
rings shows a lower ratio with respect to the in-plane
deformation modes than on the gold nanoparticles (compare
respective red and black spectra in Figure 3A). In the case of
Ami, this is less pronounced, and a C−C stretching of the
seven-membered ring appears at 782 cm−1. All SERS spectra
measured with silver nanoparticles, those excited at 532 nm
(Figure 3B) as well as those obtained at an excitation
wavelength of 785 nm (Figure S4), support the observations
made in the SEHRS spectra, which point at an interaction of
the ring system with the silver surface. As examples, the
breathing and strong C−C stretching modes of the ring at 973
and ∼1040 cm−1, respectively, are enhanced, and the band at
1207 cm−1 of one of the dibenzazepine C−C stretching modes
is quite pronounced. The SEHRS spectra and also the SERS
data observed here confirm the interaction of the molecules
with the silver surface that was reported in SERS spectra
previously.30 The larger enhancement of the in-plane modes of
the ring system than of the out-of-plane modes in our SERS
spectra obtained with the silver nanostructures is in agreement
with a proposed tilted orientation.30 It has been suggested that
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The Journal of Physical Chemistry C Article

one of the phenyl rings of Des is closer to the nanoparticle two-photon SERS spectra of the drug molecules employing
surface, while Imi interacts through the whole ring system.30 biocompatible gold nanoparticles is much more attractive with
Even though some in-plane vibrational modes of the rings, potential applications in live cells or tissues in mind. Therefore,
such as the band at 560 cm−1, are strongly enhanced in the we also collected SERS spectra at an excitation wavelength of
SERS spectra obtained with gold nanoparticles (see e.g., Figure 785 nm under such conditions. As was shown already in the
S4), these data indicate in general significant increases in the discussion of Figure S4, SERS spectra can be measured with the
enhancement of some out-of-plane vibrations, together with gold nanostructures at an excitation wavelength of 785 nm, and
additional vibrations from the aliphatic chain. The former all experiments occur in a Raman microscpectroscopic setup,
becomes evident specifically from the SERS data obtained at enabling the selection and mapping of different microscopic
532 nm (Figure 3B) and also at 785 nm (Figure S4). As an structures, such as live cells.29,32 Using these excitation
example, SERS spectra excited at 785 nm display pronounced conditions, we performed experiments using gold nanoparticles
signals at ∼760 and ∼790 cm−1, assigned to the out-of-plane in environments that are relevant for experiments with cell
deformation of the phenyl rings with a contribution of wagging cultures.
of the amino group in the aliphatic tail30 (Figure S4). Even though the spectra excited at 785 nm of Figure S4 or
Furthermore, enhancement of the band of the phenyl rings Figures 4−6 differ qualitatively from the spectra measured at an
twisting at ∼620 cm−130 is in accordance with the strengthening
of out-of-plane modes in the flat orientation of the molecules.
The strong contributions from the aliphatic chain when the
TCA molecules interact with the gold surface are very obvious
in the SEHRS spectra (Figure 3A). This is suggested by the
wealth of deformation modes of CH2 and CH3 groups,
including those at 1487, 1475, 1386, and also ∼1330 cm−1.
Interaction of all molecules with the gold surfaces via the
methylaminopropyl side chain is visible in the SERS spectra as
well. The SERS spectra of all TCA molecules on gold
contribute information on the N−C deformation vibrations
of the methylaminopropyl side chain at 846 and 812 cm−1
(Figure 3B), as well as on vibrational modes of the CH2 groups
other than those found in SEHRS, at 1462, ∼1440, 1363, and
also at 1335 and 1030 cm−1 (Figures 3B and S4). According to
the presented data, the Des and Imi molecules interact with the
gold nanostructure with both the ring system and aliphatic tail.
Because of the rigidity of the Ami molecule that is caused by
the CC double bond in the aliphatic chain, the spectra show
an interaction of the dibenzazepine system with the gold
surface different from Des and Imi, as evidenced, e.g., by the
relative intensities of the C−C modes at 1206 and 1623 cm−1
(first traces in Figures 3A and S3A). An interaction with the
surface via the phenyl rings would force the molecules into a
tilted position. This is supported by the presence of both out-
of-plane and in-plane modes in the spectrum.48 In such a tilted
orientation, an additional interaction through the amino group
in the aliphatic chain can take place.
Both SEHRS and SERS data indicate that the molecular
orientation on the surface compared to the interaction with the
silver is very different. The decrease in the intensity of the in-
plane vibration of the ring system in the spectra on the gold
nanostructures suggests a change in the orientation from tilted
to the flat in Des and Imi.50 Furthermore, the increase in the
intensity of the mode from aliphatic tail suggests closeness of
this part of the molecule to the surface of gold nanoparticles. Figure 4. SERS spectra of the antidepressant desipramine obtained
The different interaction of the gold surface with the ring using gold nanostructures with a diameter of 30 nm (Au30) and a
moiety and the important function of the alkyl chain in the diameter of 60 nm (Au60). Excitation wavelength, 785 nm; excitation
interaction could render part of the ring structure more easily intensity, 2.0 × 105 W cm−2; acquisition time, 1 s; concentration,
accessible for interaction with potential biological targets, for 9 × 10−7 M; scale bars, 20 cps.
example with lipid membranes.53
In Vitro Spectra in Biological Experiments. In the excitation wavelength of 532 nm (Figure 3B, first, third, and
discussion and interpretation of the near-infrared (NIR) fifth traces) because of known effects in Raman experiments at
(1064 nm) excited SEHRS spectra, the SERS data excited at different wavelengths, such as varied detector and optics
the corresponding second harmonic wavelength (532 nm) were sensitivity or ν4-dependence of the Raman signal, they appear
very useful because of the same frequency range of the hyper specifically favorable with respect to the SERS signals. This is
Raman and Raman light (Figure 3). Nevertheless, the due to the plasmonic properties of the gold nanoaggregates in
possibility to use NIR excitation to obtain both one- and the presence of the molecules, providing high electromagnetic
22964 DOI: 10.1021/acs.jpcc.7b08026
J. Phys. Chem. C 2017, 121, 22958−22968
The Journal of Physical Chemistry C
Figure 5. SERS spectra of the antidepressants desipramine obtained
using gold nanostructures without the DMEM-FCS and in the
presence of DMEM-FCS. Excitation wavelength, 785 nm; excitation
intensity, 2.0 × 105 W cm−2; acquisition time, 1 s; concentration of
desipramine, 9 × 10−7 M; scale bars, 50 cps. The bands highlighted in
red show slight intensity variations and are discussed in the text.
enhancement and low reabsorption of the SERS light.39,54−57
The varied relative intensities and greater wealth of bands
specifically in the frequency region below 900 cm−1 indicates
that both electromagnetic and chemical enhancement deter-
mine the spectral signatures.
The spectra were measured at several concentrations, ranging
from 10−4 M to 10−7 M, all yielding very similar spectral
fingerprints. We estimate that full coverage of the nanoparticle’s
surface is achieved around 10−6 M concentration. Furthermore,
spectra were obtained with the different batches of gold
nanoparticles, possessing citrate as stabilizing species but of
varying size. As an example, Figure 4 shows the spectra of
desipramine obtained with gold nanoparticles of a diameter of
30 and 60 nm, respectively. They indicate very small differences
in two twisting vibrations of the rings with a slight relative
increase of the band at 703 cm−1 on the larger nanoparticles
(Figure 4, upper spectrum).
An important step is to assess the stability of the
nanoparticles and the spectral signatures in the biological
conditions and interaction with components of potential
delivery media. Components present in cell culture growth
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Article
Figure 6. SERS spectra of the antidepressant desipramine obtained
using gold nanostructures at pH 3.4, pH 4.4, and pH 6.0. Excitation
wavelength, 785 nm; excitation intensity, 2.0 × 105 W cm−2;
acquisition time, 1 s; concentration of desipramine, 9 × 10−7 M;
scale bars, 30 cps. The bands highlighted in red show slight intensity
variations and are discussed in the text.
medium, used for delivery of the nanoparticles-drug system,
have an effect on the cellular uptake and further behavior of
nanoparticles in cells.58 Upon entering the cell culture medium,
and later inside the cellular environment, nanoparticles are
usually coated with proteins present in the surroundings that
become visible in the SERS spectra.59−61 To understand
whether components from the cell culture media may influence
nanoparticle−drug interaction, spectra were obtained in the
presence of a typical cell culture medium DMEM and FCS,
which is an important constituent of cell culture media. UV−vis
spectra of the drugs adsorbed on gold nanoparticles were
obtained in the cell culture medium, and Figure S5 shows the
example of desipramine. As discussed, Des causes an
aggregation of the nanoparticles (Figure 2D). After addition
of the DMEM- FCS to the nanoparticles-drug system, a slight
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J. Phys. Chem. C 2017, 121, 22958−22968
The Journal of Physical Chemistry C
blue shift of the extended plasmon band is visible, indicating
interaction of the molecules from the culture medium with the
nanoaggregates surface. The SERS spectrum of Des in the
presence and absence of DMEM-FCS is shown in Figure 5. It
does not exhibit great changes. Nevertheless, some smaller
changes are noted. The relative intensity of the bands at 760
and 791 cm−1 is changed. The slight decrease of the signal
assigned to the twisting of the phenyl ring coupled with
wagging of NH in aliphatic chain at 791 cm−1 could indicate a
slightly more upright position in the presence of potentially
coadsorbed components from the cell culture medium.
Furthermore, the relative intensity in the band at 1538 cm−1
decreases in the presence of DMEM-FCS. Nevertheless, neither
the main vibrational signature of desipramine including relative
intensities nor the overall enhancement is influenced in the
presence of the culture medium. Considering the strong affinity
that some of the components of DMEM-FCS have toward the
surface of gold nanoparticles,62,63 we can conclude that
interaction between the TCA molecules and nanoparticles is
very stable.
In biological surroundings, nanoparticle-based drug delivery
systems can encounter very different pH values, depending on
the route of delivery and uptake into tissues and cells. In Figure
6, the SERS spectra of Des at different pH values are shown.
The characteristic spectrum is not influenced; nevertheless,
small changes in some relative intensities occur. The band at
535 cm−1 decreases when pH decreases from pH 6 to pH 3.4.
At the same time, the in-plane bending mode of the
dibenzazepine ring at 585 cm−1 intensifies (compare top trace
in Figure 6 with middle and bottom traces), while the ring
twisting mode at 710 cm−1/703 cm−1 becomes weaker.
Changes in these vibrational modes indicate a slight
reorientation of the ring systems with respect to the gold
nanoparticle surface, when pH is lowered. Similarly, the band at
772 cm−1, assigned to ring deformation modes of the phenyl
rings and C−C stretching of the seven-membered ring,
disappears, maybe pointing toward a more tilted orientation
when pH decreases. Additionally, smaller influences on bands
at 1534 and 1570 cm−1 can be seen. Because the TCA are
molecules with a very high pKa value (∼9.5), we do not expect
that the change in pH values has a direct influence on the
protonation/deprotonation of molecules themselves but rather
indirectly on the interaction by protonation and deprotonation
of the stabilizing species and interaction with the citrate
molecules on the nanoparticle surfaces.
The robustness of the spectral signature nanoparticle−drug
system to changing pH suggests that studies inside the
endosomal system, where pH lowers significantly over time,
are feasible. Previously reported pH values of the early
endosomes are around 6, and during the process of maturation,
pH becomes more acidic and reaches as low as 4 in the
lysosome.64 Furthermore, several pathologies are associated
with very low pH values in the tissues.65
The fact that the TCA−gold nanostructure assemblies are so
stable could inspire new kinds of multifunctional nanocarriers
for TCA molecules. In general, nanoparticle-based drug carriers
can offer many advantages for targeted delivery including
optical imaging and monitoring possibilities. Our results here
show that the characterization of the interaction of a gold−
TCA drug delivery system would in principle be feasible. The
function of SERS and SEHRS therein would lie in a sensitive
probing of the molecules attached to the surface and possibly
also the cellular environment and its response to the delivered
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Article
drug.66 Obtained SERS and SEHRS spectra here, especially
under the biological conditions, may be used for further
investigation of nanoparticle-based drug delivery platforms for
tricyclic antidepressants.
■ CONCLUSIONS
We have reported the acquisition of nonresonant SEHRS and
SERS spectra of the three TCA molecules amitriptyline,
desipramine, and imipramine, exploiting the interaction of the
molecules with gold nanostructures, and compare them to the
interaction with silver nanostructures. The SEHRS spectra of
the TCA molecules differ greatly from the SERS spectra
because of the different selection rules of the one- and two-
photon excited Raman processes. Specifically, they show several
characteristics of infrared-active vibrations.
We find that Raman and hyper Raman active vibrational
modes can be combined for the comprehensive analysis of the
molecules’ interaction with the nanostructures. From the
SEHRS spectra excited at 1064 nm and SERS spectra obtained
at two different excitation wavelengths (532 and 785 nm), we
infer that the molecules interact with the silver nanostructures
mainly via their ring moiety and less intensely with the alkyl
chain, which is in line with previous findings by SERS.30 In
contrast, as revealed from the SEHRS and SERS spectra
obtained on biocompatible gold nanostructures, in all three
molecules, the methylaminopropyl side chain plays a very
important role in the interaction with the gold, along with parts
of the ring system. This is very different from the interaction
with the silver nanostructures.
The NIR excited SERS spectra of the TCA−gold nano-
particles are greatly invariant with respect to changes in TCA
concentration and size of the biocompatible gold nanostruc-
tures. They show remarkable stability in the presence of cell
culture media and upon decrease of pH in the typical ranges of
pH values in late endosomal structures. These findings suggest
the combined one- and two-photon vibrational spectroscopic
approach as a tool for monitoring and characterization of new
nanoparticle-based drug delivery platforms and their interaction
in real biological environments.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jpcc.7b08026.
UV−vis absorbance spectra of the gold and silver
nanostructures (Figure S1); Raman spectra of tricyclic
antidepressants, desipramine, imipramine, and amitripty-
line (Figure S2); SEHRS and SERS spectra of the
antidepressants in the range 1400−1700 cm−1 (Figure
S3); SERS spectra of the antidepressants imipramine,
desipramine, and amitriptyline obtained using silver and
gold nanostructures with 785 nm excitation wavelength
(Figure S4); UV−vis absorbance spectra of the gold
nanostructure in the presence of desipramine and
DMEM-FCS (Figure S5) (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: janina.kneipp@chemie.hu-berlin.de.
ORCID
Janina Kneipp: 0000-0001-8542-6331
DOI: 10.1021/acs.jpcc.7b08026
J. Phys. Chem. C 2017, 121, 22958−22968
The Journal of Physical Chemistry C
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We thank Harald Kneipp for valuable discussions and support
in setting up experiments. V.Ž . and Z.H. are grateful for
fellowships provided by DFG GSC 1013 SALSA; F.M.
acknowledges funding by a Chemiefonds Fellowship of FCI
(Fonds der Chemischen Industrie), C.A. by DFG Grant No.
AR376/12-1, and J.K. by ERC Grant No. 259432 MULTI-
BIOPHOT.
■
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DOI: 10.1021/acs.jpcc.7b08026
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