Peptide-Driven Targeted Drug-Delivery System Comprising Turn-On Near-Infrared Fluorescent Xanthene–Cyanine Reporter for Real-Time Monitoring of Drug Release

Article abstract of DOI:10.1002/cmdc.201900464

Targeted drug delivery (TDD) is an efficient strategy for cancer treatment. However, the real-time monitoring of drug delivery is still challenging because of a pronounced lack of TDD systems capable of providing a near-infrared (NIR) fluorescence signal for the detection of drug-release events. Herein, a new TDD system, comprising a turn-on NIR fluorescent reporter attached to an anticancer drug and targeting peptide, is reported. This system provides both TDD and NIR fluorescence monitoring of drug-release events in target tissue. In this TDD system, a new carboxy-derivatized xanthene–cyanine (XCy) dye is attached to an anticancer drug, chlorambucil (CLB), through a hydrolytically cleavable ester linker and coupled to a targeting peptide, octreotide amide (OCTA), which is specific to somatostatin receptors SSTR-2 and STTR-5 overexpressed on many tumor cells. This OCTA-G-XCy-CLB (G: γ-aminobutyric acid) conjugate exhibits no detectable fluorescence, whereas, upon the hydrolytic cleavage of the ester linker, a bright NIR fluorescence appears at λ≈710 nm; this signals release of the drug. Real-time TDD monitoring is demonstrated for the example of the human pancreatic cancer cell line overexpressing SSTR-2 and STTR-5, in comparison with the noncancerous Chinese hamster ovary cell line, which contains a reduced number of these receptors.

Full text of DOI:10.1002/cmdc.201900464

Accepted Article
Title: Peptide-Driven Targeted Drug Delivery System Comprising Turn-
On NIR Fluorescent Xanthene-Cyanine Reporter for Real Time
Monitoring of Drug Release
Authors: T. M. Ebaston, Alex Rozovsky, Alisa Zaporozhets, Andrii
Bazylevich, Helena Tuchinsky, Vered Marks, Gary Gellerman,
and Leonid Patsenker
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201900464
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900464
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ChemMedChem
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FULL PAPER
Peptide-Driven Targeted Drug Delivery System Comprising Turn-
On NIR Fluorescent Xanthene-Cyanine Reporter for Real Time
Monitoring of Drug Release
[
a]
[a]
[a]
[a]
[b]
T. M. Ebaston, Alex Rozovsky, Alisa Zaporozhets, Andrii Bazylevich, Helena Tuchinsky, Vered
[
a]
[a]
[a]
Marks, Gary Gellerman, and Leonid D. Patsenker*
[
a]
Mr. T. M. Ebaston, Mr. Alex Rozovsky, Ms. Alisa Zaporozhets, Mr. Andrii Bazylevich, Dr. Vered Marks, Prof. Gary Gellerman, Prof. Leonid D. Patsenker
Department of Chemical Sciences
Ariel University
Ariel, 40700 (Israel).
E-mail: leonidpa@ariel.ac.il
ORCID: 0000-0002-5541-349X
Ms. Helena Tuchinsky
Department of Molecular Biology
Ariel University
[
b]
Ariel, 40700 (Israel).
Supporting information for this article is given via a link at the end of the document.
Abstract: Targeted drug delivery (TDD) is an efficient strategy for
cancer treatment. However, the real time monitoring of drug delivery
is still challenging because of a pronounced lack of TDD systems
capable of providing near-infrared (NIR) fluorescence signal for
detection of drug release events. Herein, we first report on a novel
TDD system comprising turn-on NIR fluorescent reporter attached to
an anticancer drug and targeting peptide. This system provides both
targeted drug delivery and NIR fluorescence monitoring of drug
release events in target tissue. In this TDD system, a new, carboxy-
derivatized xanthene-cyanine dye (XCy) is attached to an anticancer
drug chlorambucil (CLB) via a hydrolytically cleavable ester linker
and coupled to a targeting peptide octreotide amide (OCTA), which
is specific to somatostatin receptors SSTR-2 and STTR-5
overexpressed on many tumor cells. This OCTA-G-XCy-CLB
conjugate exhibits no detectable fluorescence, while upon the
hydrolytic cleavage of the ester linker a bright NIR fluorescence
appears at about 710 nm signaling the drug release. The real time
TDD monitoring is demonstrated in the example of human
pancreatic cancer (Panc-1) cell line overexpressing SSTR-2 and
STTR-5 in comparison to non-cancerous Chinese hamster ovary
TDD systems being additionally equipped with a turn-on
fluorescent reporter sensitive to drug release, open the way to
[
4,5]
real time monitoring of drug delivery in vitro and in vivo.
The
non-fluorescent reporter bound to the drug by means of a
cleavable linker, predominantly degradable in tumor, becomes
fluorescent upon the drug release and thus provide highly
important information on drug distribution and drug release
[
6,7]
kinetics.
Turn-on fluorescent dyes currently used in TDD systems
[
8 ]
[ 9 ]
[ 10 ]
such as coumarin,
fluorescein
BODIPY,
1,8-naphthalimide,
and
[
11]
suffer from short wavelength emission, which is
not suitable for drug release monitoring in vivo. Longer
wavelength reporters absorbing and emitting within the near-
infrared (NIR) spectral region (~650–900 nm) are advantageous
for bioimaging due to deeper light penetration in body,
minimized photo-damage to tissue and lower interference from
[
12 ]
auto-absorption and auto-fluorescence of biomolecules.
However, to the best of our knowledge, the turn-on NIR
fluorescent reporters have never been utilized in peptide-driven
TDD systems.
(
CHO) cell line containing a reduced number of these receptors.
On the other hand, turn-on NIR fluorescent xanthene-
cyanine dyes were recently designed for spectrofluorometric and
[
13 ]
[
14 ]
imaging based detection of peroxynitrite,
fibroblasts,
[
15 , 16 ]
[ 17 ]
[
18 ]
aminopeptidase,
nitroxyl,
hydrogen polysulfide,
Introduction
[
19
]
[
20
]
carboxylesterase,
hypochlorous
These dyes were proposed also for keloid
acid,
and
[
21, 22]
glutathionine.
Targeted drug delivery (TDD) systems comprising an anticancer
drug conjugated with a cancer-specific carrier such as a peptide,
antibody or nanoparticle have gained considerable attention for
their applications in the area of cancer therapy, since they
enhance the selective removal of cancer cells without affecting
the normal tissues and thus significantly improve the therapeutic
[23]
[24]
diagnosis and chemo-photodynamic therapy. However, the
reported xanthene-cyanines did not contain reactive
functionalities for binding to a targeting carrier and until now
none of them were utilized for fluorescence TDD monitoring.
In this work, we first employ the xanthene-cyanine
fluorophore as a turn-on NIR fluorescent reporter in a peptide-
targeted delivery of anticancer drug, chlorambucil (CLB). CLB is
[
1 ]
efficacy and reduce side-effects of anticancer medication.
Targeting peptide carriers have several advantages over
antibodies and nanoparticles due to their small size, which
provides better cell penetration, higher biological activity per
mass and less chance of affecting the immune system
Peptides are also favorable owing to their natural degradability,
lower manufacturing costs, better stability upon storage, ease of
an FDA approved chemotherapy medication performing as a
[25]
DNA alkylator and used to treat leukemia and lymphoma.
As
a cancer-specific peptide, octreotide amide (OCTA) was chosen.
OCTA is an analog of cyclic, S-S bridged octapeptide
[
2 ]
.
(sandostatin) which targets overexpressed somatostatin
receptors SSTR-2 and STTR-5 on tumor cells and internalizes
[
3]
chemical modification, and controllable linkage.
[26]
into the cell by SSTR mediated endocytosis.
1
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ChemMedChem
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FULL PAPER
O
N
NH
2
ester linker. Then, the obtained TDD system was removed from
the resin by TFA.
H
2
N
H
HO
N
O
H
N
O
To estimate the effect of OCTA on the spectral properties
and drug release rate, we also synthesized and investigated the
dye–drug conjugate, XCy-CLB, containing no OCTA. The
synthesis consisted in pre-activation of CLB with thionyl chloride
in DCM followed by condensation of the obtained CLB
carboxylic acid chloride with the XCy hydroxyl group (Scheme
OH
Switchable
NIR reporter
XCy
O
O
O
N
H
O
NH
NH
S
S
GABA (G)
HN
O
O
N
H
H
NH
N
O
N
O
Hydrolytically
cleavable
ester linker
O
H
O
Drug
CLB
1b).
Targetting peptide octreotide
Both conjugates were obtained in quantities required for our
research and the synthetic procedures can be scaled up to
produce larger amounts.
amide (OCTA)
Cl
N
Cl
Molecular structure
Figure 1. The developed TDD system OCTA-G-XCy-CLB.
The structure confirmation and a full assignment of the
signals for XCy and XCy-CLB were performed by the 1D NMR
1
13
To realize this strategy, we modified the xanthene-cyanine
dye with a reactive carboxylic functionality (dye XCy) that was
further utilized for binding this dye to OCTA via the amino acid
GABA (G) linker (Figure 1). This linker increasing the peptide–
dye distance was introduced to avoid overlapping of the dye with
(
H and C) and 2D NMR experiments using the coupling
1
1
1
13
constant correlation ( H- H COSY, H- C-HMQC and HMBC).
The full atom numbering is shown in Figure S1 while the
numbering of the most important atoms discussed below is
shown in Scheme 1b. The NMR spectra are presented in
Figures S2-S13.
[
27]
the receptor recognition pharmacophore of the peptide carrier.
The obtained OCTA-G-XCy conjugate was then coupled to
anticancer drug, chlorambucil (CLB) to form novel TDD system,
OCTA-G-XCy-CLB, that was demonstrated to enable
fluorescence monitoring of drug release within the NIR spectral
region. CLB was bound to the XCy by means of a hydrolytically
cleavable ester bond which is known to be predominantly
hydrolyzed in tumor under physiological conditions in the
The 1D NMR of XCy-CLB compared to XCy shows the
presence of the CLB signals. The fact that the CLB is connected
to XCy in the XCy-CLB conjugate is confirmed by the chemical
17
18
14
shifts of the o- and p-carbon atoms (C , C and C ) and
17
18
o-hydrogens (H , H ) of the xanthene moiety, which are
different from those for XCy (Scheme 1b). All the mentioned
signals are deshielded in XCy-CLB, which is more likely due to
the electron withdrawing effect of the carbonyl group. The
[
28 ]
presence of esterases . At the same time, ester linker is
proven to be stable enough in blood stream upon target delivery
of these agents to cancer tissue and used therefore for
conjugation of various anticancer agents such as camptothecin
4
5
hydrogen coupling constant for the cyanine CH =CH double
bond connecting the indolenine and the xanthene fragments
3
(
J
H-H ~ 15 Hz for XCy and XCy-CLB) indicates that these
[
29]
and doxorubicin. We assume that CLB delivered by peptide
conjugate OCTA-G-XCy-CLB will show cytotoxic effect also in
solid tumor cell lines, similar to CLB linked to cilengitide
moieties are in the trans conformation.
(
a)
SPPS
, 2, 3
1
S
S
OCTA
[
30]
NH2
carrier.
Thr - Cys - Thr - Lys - DTrp - Phe - Cys - DPhe - GABA - NH2
Octriotide amide - GABA (OCTA-G)
Rink Amide resin
1
2
3
. Fmoc removal, 20 % Piperidin in NMP
HATU, DIPEA
NMP
HOOC
. Coupling, PyBOP (2 eq), AA (2 eq), DIPEA (8 eq), 1.5 h
(H
2
C)
N
5
. Cyclization step, I
2
(10 eq.) in DMF:H
2
O (4:1), 2h
Results and Discussion
H
N
H
H
OCTA
N N
G XCy
O
Synthesis
BTC, Collidine, CLB
DCE
XCy
OH
K
2
CO
3
/ CH
3
CN
O
CLB
50 C, 8 h
o
HO
H
H
H
The new dye, XCy, containing a reactive carboxylic group for
binding to the targeting carrier was synthesized similarly to the
N
OCTA
N
G
N
XCy
O
OH
TFA
Cl
[
17]
recently proposed method. The synthetic strategy consisted of
the straightforward nucleophilic substitution of the chlorine atom
in pre-synthesized dye, Cy, with resorcinol to eliminate the
indolenine moiety by a retro-Knovenagel reaction followed by
cyclization and dehydration to the aimed dye, XCy (Scheme 1a).
N
N
(CH
O
H
H
(CH2)5
2 5
)
H N OCTA
2
N
G
N
XCy
O
CLB
COOH
COOH
Cy
OCTA-G-XCy-CLB
(
b) HOOC
(H C)
HOOC
(
2
5
1
CH
N
1
2
)
5
DCM, DMAP
4
[
31]
N
4
o
A mechanism of this reaction was proposed elsewhere.
50 C, 6 h
2
3
2
5
5
14 (p)
O
3
The TDD system, OCTA-G-XCy-CLB, was synthesized
through solid phase peptide synthesis. The peptide sequence
was built upon a solid support (rink amide resin) using Fmoc
chemistry protocol. After the synthesis of the linear peptide, the
N-terminal Fmoc group was removed and the peptide was
O
14 (p)
Cl
Cl
O
1
7 (o)
18 (o)
Cl
17(o)
1
8 (o)
O
O
XCy
N
HO
O
Cl
Cl
Cl
OH
N
N
[
32]
cyclized by the treatment with excess I
2
in DMF.
1) SOCl , DCM, 0 C, 3 h
o
2
XCy-CLB
CLB
3
2) Et N
The obtained targeting peptide, OCTA, immobilized on a
rink amide resin was attached to XCy. Thereafter, the CLB
carboxyl function was activated by treatment with BTC and
collidine in DCE and coupled to the XCy hydroxyl group via an
Cl
Scheme 1. Synthesis of OCTA-G-XCy-CLB (a) and XCy-CLB (b).
2
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1
2
Furthermore, it was found that the indolenine N =C double
increases and a fluorescence band simultaneously appears at
~710 nm (λex = 650 nm). The rate of the CLB cleavage was
found to be much higher in CM compared to PB.
4
5
bond is located in the s-trans form to the CH =CH bond. Thus,
in the 2D H- H NOESY experiment there is a cross correlation
1
1
between the indolenine 3-methyl groups (δ = 1.81 ppm and
Based on the time-dependent absorption and emission
spectra, corresponding ester bond cleavage profiles were
obtained (Figure 3) and the drug release rates were quantified
by half-lives (τ1/2). The cleavage profiles and half-lives obtained
spectrophotometrically and spectrofluorimetrically almost
coincide. The CLB release from the XCy-CLB platform (Figure 3)
in CM (τ1/2 ~ 40 min) was found to be about 12-fold faster
compared to that in PB (τ1/2 ~ 8 h). The release of CLB from
OCTA-G-XCy-CLB in CM (τ1/2 ~ 30 min) is 4-fold faster than that
in PB τ1/2 ~2.5 h. Thus, due to the presence of esterases in CM,
the drug release kinetics for both XCy-CLB and OCTA-G-XCy-
CLB in CM is much faster compared to PB. The obtained half-
lives of the ester bond cleavage in these media were found to be
5
1.82 ppm for XCy and XCy-CLB, respectively) and the H atom
(δ = 8.73 ppm and δ = 8.78 ppm for XCy and XCy-CLB). This
conformation is also supported by a weak cross peak between
the indolenine 3-methyl hydrogens and the xanthene hydrogen
17
H
(δ = 7.28 ppm δ = 6.86 ppm for XCy and XCy-CLB) (Figure
S7, S13). The formation of the ester linkage between XCy and
CLB in XCy-CLB was also proved by considering the C=O and
OH signals in the FTIR spectra (Figure S14).
Spectral properties and drug release kinetics
The absorption and emission spectra of XCy, XCy-CLB and
OCTA-G-XCy-CLB were investigated in two different solvent
systems commonly used in biological experiments, 10 mM
phosphate buffer pH 7.4 (PB) and RPMI-1640 cell culture
[
35]
in good agreement with recently reported data.
Linkage of
OCTA to the conjugate accelerates the drug release by a factor
of 6 in PB but only 1.3 in CM.
[
33, 34]
medium pH 7.4 (CM) contained different esterases
that
The effect of OCTA on the CLB cleavage rate can be
presumably explained by its influence on the aggregation of
these conjugates. The degree of aggregation can be estimated
by the intensity of the aggregation band appeared on the short-
wavelength slope of the absorption spectrum. It can be seen
from the Figure. S15, that aggregation of OCTA-G-XCy-CLB
(curve E) is less pronounced compared to XCy-CLB in PB
(curve C). As a result, the cleavable ester bond in OCTA-G-XCy-
CLB is perhaps more sterically accessible to hydrolytic attack,
which results in the faster hydrolysis kinetics. In CM, the impact
of OCTA on the aggregation is much less pronounced (Figure.
accelerate the ester bond cleavage as compared to PB. The
obtained spectral characteristics are presented in Table S1.
Free dye XCy (“on” form) in PB and CM has the absorption
maximum of about 680 nm and strong NIR fluorescence at
around 710 nm (Figure S15). The absorption maximum of XCy
bound to CLB (“off” form) in both conjugates, XCy-CLB and
OCTA-G-XCy-CLB, is about 80 nm blue shifted to ~600 nm
(Figure S15) and the fluorescence is not detectable. Therefore,
hydrolytic cleavage of the ester bond in XCy-CLB and OCTA-G-
XCy-CLB to release free CLB results in dramatic increase of
fluorescence, which can be used for the drug release monitoring. S15, curves D and F) and the CLB release rate is also almost
To estimate the kinetic rate of the CLB release in the
presence (CM) and absence (PB) of esterases, the time-
dependent absorption and fluorescence spectra of XCy-CLB and
OCTA-G-XCy-CLB were measured (Figure 2). The conjugates
were taken at the same concentration (0.5 µM), incubated at
unaffected by the presence of OCTA.
Importantly, the LC/MS data indicate that the ester bond
cleavage resulting in the release of free CLB molecules occurs
much faster compared to the hydrolysis of chlorine atoms
(Figure S16).
37 °C and measured at 25 °C at certain time intervals. With
respect to time, the absorption band of these conjugates in both
solvent systems at ~600 nm decreases, a new band at ~680 nm
Figure 2. Time dependent absorption (a,c,e,g) and fluorescence (b,d,f,h) spectra of XCy-CLB (a,b,c,d) and OCTA-G-XCy-CLB (e,f,g,h) in PB (a,b,e,f) and CM
c,d,g,h). The 0.5 µM solutions of the compounds were prepared in 10 mM PB and CM (pH 7.4). Both solutions contained 10% methanol to facilitate solubility.
The solutions were filtered through a 0.45 µm PVDF filter and incubated at 37 °C. The absorption and emission spectra of the solutions were recorded at 25 ºC in
-cm quartz cells over certain time periods. λex = 650 nm.
(
1
3
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Panc-1 and CHO were incubated at 37 °C for 10 min with
10 µM OCTA-G-XCy-CLB in PBS pH 7.4 containing 3% DMSO
and washed thoroughly with PBS pH 7.4 to remove excess
conjugate. Then the fluorescence microscopy images were
taken over time (Figure 4 a, b).
The time dependent fluorescence intensities for both cell
lines gradually increase signaling the CLB release and then
achieve saturation. The movies demonstrating the increase of
the fluorescence intensity upon the drug release in Panc-1 and
CHO can be found in supporting information (Movie-1-Panc-
Figure 3. The spectrophotometrically (Abs) and spectrofluorimetrically (Fl)
estimated cleavage profiles for XCy-CLB and OCTA-G-XCy-CLB conjugates
1.avi and Movie-2-CHO.avi). The drug cleavage profiles for
(0.5 µM) in PB (a) and CM (b). The concentrations of the compounds are
0
.5 µM (pH 7.4). λex = 650 nm.
OCTA-G-XCy-CLB (Figure 4 c) indicate that the CLB release
kinetics in Panc-1 is much faster (by about 6-fold) compared to
CHO. The cleavage half-life (τ1/2) in Panc-1 is about 25 min
while for CHO τ1/2 ~2.5 h.
We also compared the brightness of Panc-1 and CHO
after saturation (non-normalized integrated fluorescence
intensity obtained at the same instrument setup), which must
correlate with uptake of OCTA-G-XCy-CLB by these cells. The
saturated brightness for Panc-1 was found to be about 3.4 times
higher, which indicates better specificity of this conjugate to
Panc-1 compared to CHO.
Fluorescence imaging and real time monitoring of drug
release
In the next step, we demonstrated the performance of
OCTA-G-XCy-CLB in real time monitoring of drug delivery
kinetics in the example of human pancreatic cancer (Panc-1) cell
line overexpressing somatostatin receptors SSTR-2 and STTR-
[
36]
5
in comparison to non-cancerous Chinese hamster ovary
(CHO) cell line containing a decreased number of these
[
37]
receptors.
Figure 4. Time dependent fluorescence, bright field and overlay microscopy images of Panc-1 (a) and CHO (b) cell lines. The cells were incubated at 37 °C for
0 min with 10 µM OCTA-G-XCy-CLB in PBS pH 7.4 containing 3% DMSO and washed thoroughly with PBS pH 7.4 to remove excess conjugate. Then the
1
fluorescence and bright field images were taken over time. The drug (CLB) release profiles were estimated by relative fluorescence intensities (RFI) of selected
Panc-1 and CHO cell groups (c). The CLB cleavage half-life is τ1/2 ~25 min for Panc-1 and τ1/2 ~2.5 h for CHO. Cell growth inhibition of Panc-1 (d) and CHO (e)
pre-treated with various concentrations of OCTA-G-XCy-CLB, free CLB and free OCTA. After the treatment, the cells were incubated at 37 °C for 48 h and 72 h.
Cell inhibition was accessed using a standard XTT cell survival assay. The inhibition for each concentration point is represented by the mean ± standard error for
each independent experiment conducted in triplicate.
4
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®
Cell survival assay
with dual UV detection at 230 nm and 254 nm. A Phenomenex Gemini
0 µm RP18 110 Å, LC 250 × 21.2 mm column was used. The column
was kept at ambient temperature. Eluent A (0.1% TFA in water) and B
0.1% TFA in ACN) were used. A typical elution was a gradient from
00% A to 100% B over 35 min at a flow rate of 25 mL/min. HRMS was
1
Specificity of OCTA-G-XCy-CLB to Panc-1 and CHO cell lines
was verified also by an independent method, a standard XTT
cell survival assay. In addition, specificity and cytotoxicity of this
conjugate were compared to those of CLB and OCTA. The
obtained growth inhibition indicates that OCTA-G-XCy-CLB
preferably targets specific SSTRs overexpressed on Panc-1 with
elevated growth inhibition compared to CHO (Figure 4 d, e). The
inhibition percentage of Panc-1 was found to be about 3-fold
higher than that for CHO, which is consistent with the uptake
level estimated by the saturated brightness (3.4-fold). Notably,
neither free OCTA, nor free CLB exhibited detectible toxicity in
both cell lines, which is in agreement with previous discovery
showing that CLB in a free form is not efficient against targeted
receptor deficient cell line while upon conjugation to targeting
(
1
measured in the ESI positive mode using an Agilent 6550 iFunnel Q-TOF
LC/MS. IR spectra were recorded for the solid samples using a Bruker
a platinum diamond
Attenuated Total Reflectance (ATR) module. The spectral resolution was
FTIR ALPHA II spectrometer equipped with
–
1
set to 2 cm and 16 scans were taken per measurement.
Synthesis of Cy dye: Cy dye was synthesized according to the literature
[
39]
procedure. Yield 66%.
Synthesis of XCy dye: Potassium carbonate (276 mg, 2 mmol) and
resorcinol (220 mg, 2 mmol) were dissolved in acetonitrile (20 mL) and
stirred for 15 min under N
a solution of Cy (683 mg, 1 mmol) in acetonitrile (15 mL) and stirred for
h at 50 °C. The reaction was monitored by TLC. After the reaction was
2
atmosphere. The above mixture was added to
[
38]
peptide.
8
complete, the solvent was evaporated under reduced pressure and the
crude product was column purified (silica gel 70-230 mesh, DCM—
Conclusions
methanol, 90:10, v.v.). The XCy dye (295 mg, 61% yield) was obtained
1
as a blue solid. H NMR for XCy (400 MHz, CD
3
OD, Figure S2) δ (ppm):
5
24
8
.731 (d, J = 14.7 Hz, 1 H, H ), 7.64 (d, J = 7.4 Hz, 1 H, H ), 7.51 (m, 2
We have developed the new cancer targeted drug delivery
26
27
25
13
16
H, H , H ), 7.416 (m, 1 H, H ), 7.43 (s, 1 H, H ), 7.42 (m, 1 H, H ),
6
(TDD) system, OCTA-G-XCy-CLB, comprising the turn-on NIR
17
18
4
.86 (s, 1 H, H ), 6.85 (m, 1 H, H ), 6.45 (d, J = 14.7 Hz, 1 H, H ), 4.33
fluorescent xanthene-cyanine reporter, XCy, attached to the
SSTR-2 and STTR-5 targeting peptide octreotide amide (OCTA)
and anticancer drug chlorambucil (CLB). Synthetic approach to
this conjugate was developed and the conjugate was obtained at
reasonable yield.
The fluorescence imaging and cytotoxicity assays
performed in Panc-1 cancer cell line overexpressing
somatostatin receptors SSTR-2 and SSTR-5 and non-cancerous
CHO cell line, containing a reduced number of these receptors,
demonstrate that OCTA-G-XCy-CLB can be employed as a
potent and selective TDD system, enabling real time monitoring
of drug release kinetics in target tissues.
29
9
7
(
t, J =7.3 Hz, 2H, H ), 2.78 (m, 2 H, H ), 2.72 (m, 2 H, H ), 2.3 (t, J =
33
8
30
21
22
7
H
Hz, 2 H, H ), 1.95 (m, 4 H, H , H ), 1.8 (s, 6 H, H , H ), 1.72 (m, 2 H,
32
31 13
), 1.53 (m, 2 H, H ). C NMR (100 MHz, CD OD, Figure S3), 178.5
3
34
2
15
10
19
5
(C ), 178.36 (C ), 163.93 (C ), 163.66 (C ), 156.24 (C ), 146.54 (C ),
143.22 (C ), 143.06 (C ), 136.39 (C ), 130.46 (C ), 130.18 (C ),
1
1
28
23
13
16
26
2
5
11
24
14
18
28.02 (C ), 127.48 (C ), 123.76 (C ), 116.35 (C ), 116.02 (C ),
6
27
4
17
3
15.67 (C ), 113.58 (C ), 103.87 (C ), 102.96 (C ), 51.74 (C ), 45.88
29
33
9
8
21
22
31
(
C ), 35.54 (C ), 29.95 (C ), 28.40 (C ), 28.13 (C , C ), 27.43 (C ),
3
2
7
30
2
5.97 (C ), 25.10 (C ), 21.72 (C ). MS of XCy: calculated 484.2,
+
31 4
C H34NO , found LC-MS: m/z 484.0 (Figure S17).
Synthesis of XCy-CLB conjugate: Chlorambucil (CLB, 121 mg,
.4 mmol) and SOCl (58 µL, 0.8 mmol) were stirred in DCM (15 mL)
0
2
The developed OCTA-G-XCy-CLB system can be
considered a promising model structure for noninvasive NIR
fluorescence monitoring of targeted drug delivery. We anticipate
that this system can be modified in future with other anticancer
drugs, targeting carriers and cleavable linkers.
under N2 atmosphere at 0 °C for 3 h. Then triethylamine (167 µL,
1.2 mmol) was added to the mixture via syringe. A mixture of XCy
(
100 mg, 0.20 mmol) and DMAP (24 mg, 0.20 mmol) in DCM (20 mL)
was added via a syringe and stirred at 50 °C for 6 h. The reaction was
monitored by TLC. The solvent was evaporated under reduced pressure
and the crude product was purified by using silica gel column
chromatography (DCM—methanol, 90:10, v.v.). The XCy-CLB conjugate
1
(
93 mg, 60% yield) was obtained as a blue solid. H NMR of compound
Experimental Section
3
XCy-CLB (400 MHz, CD OD, Figure S8) δ (ppm): 8.78 (d, J = 15 Hz, 1 H,
H ), 7.67 (d, J = 7.4 Hz, 1 H, H ), 7.61 (d, J = 7.9 Hz, 1 H, H ), 7.56 (m,
5
24
27
2
6
16
25
13
Reagents and general methods: Protected amino acids, resin and
coupling reagents were purchased from Tzamal d-Chem Laboratories,
Ltd. All other chemicals were supplied by Alfa Aesar Israel and Sigma-
Aldrich. Solvents were purchased from Bio-Lab Israel and used as is.
Chemical reactions were monitored by TLC (Silica gel 60 F-254, Merck).
1 H, H ), 7.51 (m, 1 H, H ), 7.49 (m, 1 H, H ), 7.32 (s, 1 H, H ), 7.28 (s,
1
7
43
44
18
1 H, H ), 7.12 (d, J = 8.4 Hz, 2 H, H , H ), 7.05 (bd, 1 H, H ), 6.71 (d,
45
46
4
J = 8.4 Hz, 2 H, H , H ), 6.62 (d, J = 15 Hz, 1 H, H ), 4.42 (t, J = 7.5, 2
H, H ), 3.74 (m, 4 H, H , H ), 3.64 (m, 4 H, H , H ), 2.79 (m, 2 H, H ),
2.73 (m, 2 H, H ), 2.67 (m, 2 H, H ), 2.64 (m, 2 H, H ) 2.30 (t, J = 7.3
Hz, 2 H, H ), 2.03 (m, 2 H, H ), 1.95 (m, 4 H, H , H ), 1.82 (s, 6 H, H ,
), 1.71 (m, 2 H, H ), 1.53 (m, 2 H, H ). C NMR (100 MHz, CD
Figure S9), 180.33 (C ), 177.33 (C ), 173.16 (C ), 161.62 (C ), 154.50
(C or C ), 154.45 (C or C ), 147.77 (C ), 146.19 (C ), 143.86 (C ),
142.73 (C ), 132.76 (C ), 131.36 (C or C ), 131.25 (C or C ),
29
49
50
51
52
9
7
41
39
1
13
33
40
30
8
21
H
NMR and
Bruker AvanceIII HD ( H 400 MHz and
in CD OD. The signal of the remaining non-deuterated solvent was used
as an internal standard reference of the chemical shifts ( H δ = 3.31 ppm
C
NMR spectra were measured using 400 MHz
1
13
22
32
31
13
C
100 MHz) spectrometer
H
3
OD,
2
34
37
10
3
1
15
19
19
15
5
47
23
13
28
13
11
42
11
42
and C δ=49.0 ppm). The probe was equipped with Z-axis gradients
1
1
43
44
26
25
16
24
coils. A full assignment was done using 2D experiments: COSY ( H- H
130.73 (C , C ), 130.35 (C ), 129.43 (C ), 129.04 (C ) 123.88 (C ),
121.02 (C ), 120.37 (C ), 116.02 (C ), 114.45 (C ), 113.62 (C , C ),
1
13
14
18
6
27
45
46
correlation), HMQC, HMBC ( H-
respectfully) and J-resolved. The 2D homonuclear NOESY experiments
employed 128 t increments with a dwell time of 113.6 µsec and a mixing
C
short and long correlation,
17
4
49
50
3
29
110.61 (C ), 106.49 (C ), 54.55 (C , C ), 52.44 (C ), 46.41 (C ), 41.72
51
52
41
33
39
9
8
21
(C , C ), 34.97 (C , C ), 34.36 (C ), 30.27 (C ), 28.64 (C ), 28.13 (C ,
1
22
40
31
32
7
30
time of 1.5 sec with a recycle delay of 2.5 sec. LC/MS analyses were
performed using an Agilent Technologies 1260 Infinity (LC) 6120
quadruple (MS), column Agilent SB-C18, 1.8 mm, 2.1 × 50 mm, column
temperature 50 °C, eluent water-acetonitrile (ACN) + 0.1% formic acid.
HPLC purifications were carried out on an ECOM preparative system,
C ), 27.76 (C ), 27.30 (C ), 25.73 (C ), 25.00 (C ) 21.73 (C ). MS of
XCy-CLB: calculated 769.3170,
769.3174 (Figure S18).
+
45 2 2 5
C H51Cl N O , found HRMS: m/z
5
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ChemMedChem
10.1002/cmdc.201900464
FULL PAPER
Solid phase synthesis of octreotide amide-GABA (OCTA-G): The
25 °C over time. The fluorescence excitation wavelength (λex) was
synthesis of the cyclic peptide OCTA-G was done according to the
650 nm.
[
40]
previously described procedure.
Rink Amide resin (0.65 mmol/g) was
placed in a sintered glass bottom and swelled in NMP by agitation
overnight. The Fmoc group was removed from the resin by treatment
with 20% piperidine—NMP (2 x 15 min). The completion of the Fmoc
removal was monitored by ninhydrin test (blue color). After washing the
resin with NMP (7 x 2 min), a mixture of Fmoc-Thr(Trt)-OH (2 eq.),
DIPEA (8 eq.) and PyBOP (2 eq.) in NMP was added. The reaction was
carried out for 1.5 h at room temperature. After coupling, the peptidyl
resin was washed with NMP (5 x 2 min). The completion of the reaction
was monitored by ninhydrin test (yellow color). Then a linear SPPS was
applied using standard Fmoc procedures introducing the amino acids in
the following order: Fmoc-Cys(Acm)-OH, Fmoc-Thr(Trt)-OH, Fmoc-
Lys(Boc)-OH, Fmoc-DTrp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH,
Fmoc-DPhe-OH, Fmoc-GABA-OH. All the couplings were performed in
NMP with 2-fold excess of amino acid, DIPEA (8 eq.) and PyBOP (2 eq.)
for activation. Each coupling cycle was conducted for 1.5 h. The
completion of each coupling reaction and Fmoc removal were monitored
by the ninhydrin test. After the coupling of the last amino acid, the
Cell culture: The PANC-1 and CHO cell lines were cultured in an RPMI
cell culture medium supplemented with 2 mM glutamine, 10% FBS and
1
00 IU/mL of penicillin and streptomycin. The cell culture growth medium
and all its additives were purchased from Biological Industries, Israel.
The cell cultures were grown at a 37 °C in an environment containing 6%
CO .
2
Imaging: The bright field and fluorescence images were acquired by
Photometrics CoolSNAP HQ2 camera mounted on an Olympus iX81
fluorescent microscope. The microscope was equipped with a 100 W
halogen lamp for phase contrast observations and a 120 W metal halide
discharge lamp for fluorescence imaging. For the fluorescence imaging,
a cube comprising an ET620/60x bandpass excitation filter, ET700/75m
bandpass emission filter and T66lpxr dichroic filter were used. To monitor
drug release, Panc-1 and CHO cell lines were grown in six-well culture
plates, washed with PBS (pH 7.4) two times and incubated for 10 min
with 10 µM OCT(N)-G-XCy-CLB in PBS pH 7.4 containing 3% DMSO.
After incubation, the samples were twice washed thoroughly with PBS
sequence was cyclized by I
washed with DMF—H O (4:1, v.v.) 5 x 2 min, DCM 5 x 2 min, chloroform
x 2 min, 2% ascorbic acid in DMF, DMF 5 x 2 min and N-terminus
2 2
(10 eq.) in DMF—H O (4:1, v.v.) for 2 h,
2
(
pH 7.4) to remove excess conjugate. The washed cells were
5
resuspended in PBS and the fluorescence microscopy images were
taken over time. The imaging experiments were done at 37 °C in an
environment containing 6% CO and 90% humidity. All the images were
2
taken with the same instrument settings. For the bright field imaging the
exposure time was 50 ms, gain 12 dB. For the fluorescence imaging the
exposure time was 500 ms, gain 12 dB. The fluorescence intensities
were quantified by creating a region of interest (ROI) around each group
of cells and the relative fluorescence intensity of each group was
measured using the ImageJ software.
Fmoc was deprotected using regular procedure yielding the cyclic
peptidyl residue (OCTA-G) on Rink Amide resin ready for the next
conjugation.
Synthesis of OCTA-G-XCy-CLB: A mixture of XCy (35 mg, 1.2 eq.),
HATU (35 mg, 1.5 eq.) and DIPEA (50 µL, 5 eq.) in NMP was stirred for
2
min to pre-activate the XCy carboxyl function and consequently added
to the OCTA-G immobilized on solid phase (200 mg, 1 eq.). The reaction
was carried out for 1 h at room temperature. After coupling (ninhydrin test
gave yellow color), the peptidyl resin was washed with NMP (5 x 2 min)
and DCE (5 x 2 min). Thereafter, carboxyl function of the drug CLB (37
mg, 2 eq.) was activated by treatment with BTC (26 mg, 1.5 eq.) and
collidine (45 µL, 6 eq.) in 4 mL DCE for 1 min and added to the peptidyl
resin. The reaction was carried out for 1 h. After coupling, the peptidyl
resin was washed with DCE (5 x 2 min).
Cytotoxicity test: The cytotoxicity of the investigated compounds was
determined by measuring the mitochondrial enzyme activity, using a
commercial XTT assay kit (Biological Industries, Israel). All samples were
prepared in PBS pH 7.4 contained 3% DMSO. The cells were cultured in
4
micro wells (96 well-plate) at a concentration of 5–10×10 cells/well. The
cells were washed and fresh RPMI 1640 cell medium (Biological
Industries, Israel) containing different concentrations (up to 25 µM) of the
investigated compounds were added and the cells were incubated under
Cleavage procedure for rink amide resin: OCTA-G-XCy-CLB on the
peptidyl-resin from the previous step was treated with 5 ml of TFA for
CO
medium and then a fresh RPMI cell medium was added and cultured for
8 h and 72 h. At the end of the second incubation, XTT reagent was
2
at 37 °C for 10 min. The cell cultures were washed with RPMI cell
1
.5 h. After filtration of the cleavage mixture the solvent was removed
under a gentle flow of N and then the crude product was precipitated
from Et O. The obtained crude OCTA-G-XCy-CLB was purified by
preparative HPLC and lyophilized. Overall yield 8.5% (21 mg). MS of
2
4
2
added and the cells were re-incubated for an additional 3 h. During that
time the absorbances in the wells were measured with a TECAN Infinite
M200 ELISA reader at 480 nm and 680 nm. The difference in the
absorbances measured at these two wavelengths were used for
calculating the percentage Growth Inhibition (GI) in test wells compared
to two controls: cells that were exposed to fresh RPMI cell medium and
those which were exposed to a RPMI cell medium with 3% DMSO. All
the tests were done in triplicate.
+
2 14 15 2
OCTA-G-XCy-CLB: calculated 1867.7949, C98H121Cl N O S , found
+
HRMS: m/z 1868.79 [M+H] (Figure S19). LCMS chromatogram is shown
+
in Figure S20. LCMS mass spectrum 934.7 [(M+H)/2] is shown in Figure
S21.
Absorption and fluorescence measurements: Absorption spectra
were recorded on a Jasco V-730 UV–Vis spectrophotometer and the
fluorescence spectra were taken on Edinburgh FS5 spectrofluorometer.
The absorption and fluorescence spectra were measured at 25 ºC in 1-
cm quartz cells at ~0.5 µM compound concentrations in 10 mM
phosphate buffer pH 7.4 (PB) and cell culture medium pH 7.4 (CM). Both
PB and CM contained 10% methanol to facilitate solubility of the
investigated compounds. Excitation wavelength (λex) was 650 nm. Before
measurements, all the solutions were filtered through a 0.45 µm PVDF
filter. (Cell culture medium (CM, 500 mL) contained a RPMI-1640
standard cell culture medium from Sigma-Aldrich (435 mL), 50 mL fetal
bovine serum (FBS) containing esterases, 5 mL penicillin (10,000 U/mL),
Acknowledgements
This research was supported by the Israel Scientific Foundation
(ISF), project 810/18. Leonid Patsenker is thankful to the Center
for Absorption in Science of the Ministry of Immigrant Absorption
of Israel for the financial support under the KAMEA program.
Gary Gellerman is thankful to the Gale Foundation for the
generous donation to support this research.
5
mL streptomycin (10 mg/mL), 5 mL of 200 mM glutamine, and 5.3 mg/L
phenol red indicator.)
Spectral measurements of CLB release kinetics: The XCy-CLB and
OCTA-G-XCy-CLB conjugates (0.5 µM) in PB and CM were incubated at
Conflicts of interest
3
7 °C and the absorption and fluorescence spectra were measured at
There are no conflicts to declare.
6
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ChemMedChem
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FULL PAPER
[
[
[
[
33] M. F. Montenegro, M. T. Moral-Naranjo, E. Muñoz-Delgado, F. J.
Campoy, C. J. Vidal, FEBS J. 2009, 276, 2074–2083.
Keywords: cancer targeted drug delivery • fluorescence •
peptide • turn-on NIR fluorescence • xanthene-cyanine
34] A. J. Dafferner, S. Lushchekina, P. Masson, G. Xiao, L. M. Schopfer,
O. Lockridge, Chem. Biol. Interact. 2017, 266, 17–27.
35] A. Bazylevich, L. D. Patsenker, G. Gellerman, Dye. Pigment. 2017, 139,
460–472.
References:
36] M. Li, W. Li, H. J. Kim, Q. Yao, C. Chen, W. E. Fisher, J. Surg. Res.
2
004, 119, 130–137.
[
[
[
[
[
1]
2]
3]
4]
5]
R. W. Sirianni, B. Behkam, Eds. , Targeted Drug Delivery, Springer US,
New York, NY, 2018.
[
[
[
37] C. Huang, Y. Wu, S. Chen, Chem. Biol. 2000, 7, 453–461.
38] C. Huang, Y. Wu, S. Chen, Chem. Biol. 2000, 7, 453–461.
39] S. Luo, X. Tan, S. Fang, Y. Wang, T. Liu, X. Wang, Y. Yuan, H. Sun, Q.
Qi, C. Shi, Adv. Funct. Mater. 2016, 26, 2826–2835.
R. C. Ladner, A. K. Sato, J. Gorzelany, M. De Souza, Drug Discov.
Today 2004, 9, 525–529.
J. Wang, J. J. Masehi-Lano, E. J. Chung, Biomater. Sci. 2017, 5, 1450–
[
40] B. Redko, E. Ragozin, B. Andreii, T. Helena, A. Amnon, S. Z. Talia, O.
H. Mor, K. Genady, G. Gary, Biopolymers 2015, 104, 743–752.
1
459.
R. Weinstain, E. Segal, R. Satchi-Fainaro, D. Shabat, Chem. Commun.
010, 46, 553–555.
2
M. H. Lee, A. Sharma, M. J. Chang, J. Lee, S. Son, J. L. Sessler, C.
Kang, J. S. Kim, Chem. Soc. Rev. 2018, 47, 28–52.
[
[
6]
7]
R. Langer, Nature 1998, 392, 5–10.
K. Singh, A. M. Rotaru, A. A. Beharry, ACS Chem. Biol. 2018, 13,
1785–1798.
[
[
[
[
[
8]
9]
S. Maiti, N. Park, J. H. Han, H. M. Jeon, J. H. Lee, S. Bhuniya, C. Kang,
J. S. Kim, J. Am. Chem. Soc. 2013, 135, 4567–4572.
S. Bhuniya, M. H. Lee, H. M. Jeon, J. H. Han, J. H. Lee, N. Park, S.
Maiti, C. Kang, J. S. Kim, Chem. Commun. 2013, 49, 7141–7143.
10] J. Y. Kim, J. L. Sessler, C. Kang, J. H. Han, S. Bhuniya, J. S. Kim, M. H.
Lee, J. Am. Chem. Soc. 2012, 134, 12668–12674
11] S. Bhuniya, H. Lee, S. Maiti, J. L. Sessler, K. S. Hong, E.-J. Kim, J. S.
Kim, Angew. Chemie Int. Ed. 2014, 53, 4469–4474.
.
.
12] P. J. Verveer, Ed. , Advanced Fluorescence Microscopy, Springer New
York, New York, NY, 2015.
[
[
13] J. Zhang, X. Zhen, J. Zeng, K. Pu, Anal. Chem. 2018, 90, 9301–9307.
14] Q. Miao, D. C. Yeo, C. Wiraja, J. Zhang, X. Ning, C. Xu, K. Pu, Angew.
Chemie Int. Ed. 2018, 57, 1256–1260.
[
[
[
[
[
[
[
[
[
[
[
[
[
[
15] X. He, L. Li, Y. Fang, W. Shi, X. Li, H. Ma, Chem. Sci. 2017, 8, 3479–
3
483.
16] X. He, Y. Hu, wen Shi, X. Li, H. Ma, Chem. Commun. 2017, 53, 9438–
441.
9
17] Y. Tan, R. Liu, H. Zhang, R. Peltier, Y. W. Lam, Q. Zhu, Y. Hu, H. Sun,
Sci. Rep. 2015, 5, 1–9.
18] Y. Fang, W. Chen, W. Shi, H. Li, M. Xian, H. Ma, Chem. Commun.
2
017, 53, 8759–8762.
19] D. Li, Z. Li, W. Chen, X. Yang, J. Agric. Food Chem. 2017, 65, 4209–
215.
20] J. Ma, C. Yan, Y. Li, H. Duo, Q. Li, X. Lu, Y. Guo, Chem. – A Eur. J.
019, 25, 7168–7176.
21] J.-Y. Xie, C.-Y. Li, Y.-F. Li, J. Fei, F. Xu, J. Ou-Yang, J. Liu, Anal. Chem.
016, 88, 9746–9752.
22] F. Kong, Z. Liang, D. Luan, X. Liu, K. Xu, B. Tang, Anal. Chem. 2016,
8, 6450–6456.
23] P. Cheng, J. Zhang, J. Huang, Q. Miao, C. Xu, K. Pu, Chem. Sci. 2018,
, 6340–6347.
4
2
2
8
9
24] H. W. Liu, X. X. Hu, K. Li, Y. Liu, Q. Rong, L. Zhu, L. Yuan, F. L. Qu, X.
B. Zhang, W. Tan, Chem. Sci. 2017, 8, 7689–7695.
25] L. Vidal, R. Gurion, R. Ram, P. Raanani, O. Bairey, T. Robak, A.
Gafter-Gvili, O. Shpilberg, Leuk. Lymphoma 2016, 57, 2047–2057.
26] C. R. R. Grace, J. Erchegyi, M. Samant, R. Cescato, V. Piccand, R.
Riek, J. C. Reubi, J. E. Rivier, J. Med. Chem. 2008, 51, 2676–2681.
27] Z. Uddin, X. Li, B. Jasti, Curr. Trends Biotechnol. Pharm. 2019, 13,
146–155.
28] L. Tian, Y. Yang, L. M. Wysocki, A. C. Arnold, A. Hu, B. Ravichandran,
S. M. Sternson, L. L. Looger, L. D. Lavis, Proc. Natl. Acad. Sci. 2012,
109, 4756–4761.
[
[
29] R. He, B. Finan, J. P. Mayer, R. D. DiMarchi, Molecules 2019, 24, 1855.
30] Y. Gilad, E. Noy, H. Senderowitz, A. Albeck, M. A. Firer, G. Gellerman,
Bioorg. Med. Chem. 2016, 24, 294–303.
[
[
31] L. Yuan, W. Lin, S. Zhao, W. Gao, B. Chen, L. He, S. Zhu, J. Am.
Chem. Soc. 2012, 134, 13510–13523.
32] R.-H. Mattern, S. B. Moore, T.-A. Tran, J. K. Rueter, M. Goodman,
Tetrahedron 2000, 56, 9819–9831.
7
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ChemMedChem
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Entry for the Table of Contents
Drug
Enzyme
Dye
Peptide
Cancer cells
A novel system comprising near-IR switchable reporter based on xanthene-cyanine dye (XCy) attached to anticancer drug
chlorambucil (CLB) and cancer-specific targeting peptide octreotide amide (OCTA) was developed. The system enables real time
monitoring of targeted drug delivery (TDD), demonstrated in the example of human pancreatic cancer (Panc-1) and Chinese hamster
ovary (CHO) cell lines.
8
This article is protected by copyright. All rights reserved.