Biomedicine & Pharmacotherapy 134 (2021) 111154

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Chlorin e6 embedded in phospholipid nanoparticles equipped with specific

peptides: Interaction with tumor cells with different aminopeptidase
N expression
Tatyana I. Torkhovskaya a, Lyubov V. Kostryukova a, Yulia A. Tereshkina a, *,
Elena G. Tikhonova a, Galina E. Morozevich a, Anna D. Plutinskaya b, Alexey Yu. Lupatov a,
Andrey A. Pankratov b
a
Institute of Biomedical Chemistry, Pogodinskaya str., 10, Moscow, 119121, Russia
b
MNII Them. P.A. Herzen – Branch of FSBI «NIIRTS» Ministry of Health of Russia, 2nd Botkinsky Passage, 3, Moscow, 125284, Russia

A R T I C L E I N F O A B S T R A C T

Keywords: A promising direction in Biopharmaceuticals is the development of specific peptide-based systems to improve
Phospholipid nanoparticles drug delivery. This approach may increase tumor specificity and drug penetration into the target cell. Similar
Photodynamic therapy systems have been designed for several antitumor drugs. However, for photodynamic therapy drugs, such studies
Photosensitizers
are not yet enough. Previously, we have developed a method of inclusion of chlorin e6 (Ce6), a photosensitizer
NGR-containing peptide
Cell-penetrating peptide
used in photodynamic therapy, in phospholipid nanoparticles with a diameter of up to 30 nm, and reported an
Cytotoxicity increase in its effectiveness in the experiments in vivo. In this work, we propose to modify a previously developed
delivery system for Ce6 by the addition of cell-penetrating (R7) and/or targeting NGR peptides. The interaction
of the compositions developed with HepG2 and MCF-7 tumor cells is shown. The expression of CD13 protein with
affinity to NGR on the surface of these cells has been studied using flow cytometry. The expression of this protein
on the HepG2 cells and its absence on MCF-7 was demonstrated. After incubation of tumor cells with the
resulting Ce6 compositions, we evaluated the cellular accumulation, photoinduced, and dark cytotoxicity of the
drugs. After irradiation, the highest level of cytotoxicity was observed when R7 peptide was added to the system,
either alone or in combination with NGR. In addition to R7, the NGR-motif peptide increased the internalization
of Ce6 in HepG2 cells without affecting its photodynamic activity. In this work we also discuss possible mech­
anisms of action of the cell-penetrating peptide when attached to phospholipid nanoparticles.

1. Introduction synthetic tetrapyrrole compounds linked by methylene bridges [2].

Photodynamic therapy (PDT) occupies a prominent place among
various modern approaches to cancer treatment. This method involves
the administration of special substances, photosensitizers, into the body.
Photosensitizers accumulate in the tumor, after local irradiation, they
induce the production of ROS and cause cell death [1]. Most photo­
sensitizers are different versions of porphyrin derivatives – natural and
There are several types of photosensitizers, mainly related to hemato­
porphyrins [3]. Recently, a porphyrin derivative containing three
carboxyl groups called chlorin e6 (Ce6) has been widely used in clinical
practice and biopharmaceutical research [4]. Ce6 has a pronounced
anti-tumor effect. However, Ce6 has certain limitations for clinical use,
which leads to a decrease dose and a decrease in the therapeutic effect.
These limitations include low water solubility and the adverse effects of

Abbreviations: Ce6, chlorin e6; APN, aminopeptidase N; RES, reticuloendothelial system; NGR, asparagine-glycine-arginine; FBS, fetal bovine serum; DMSO,
dimethyl sulfoxide; PDT, photodynamic therapy; ROS, reactive oxygen species; PBS, phosphate-buffered saline; NPh-Ce6, chlorin e6 embedded in phospholipid
nanoparticles; NPh-Ce6-R7, phospholipid composition of chlorin e6 with a penetrating peptide, R7; NPh-Ce6-NGR, phospholipid composition of chlorin e6 with a
targeted component, NGR; NPh-Ce6-NGR-R7, phospholipid composition of chlorin e6 with penetrating and targeted peptides.
* Corresponding author at: Institute of Biomedical Chemistry, Pogodinskaya str., 10/8, lab.261, Moscow, 119121, Russia.
E-mail addresses: torti@mail.ru (T.I. Torkhovskaya), lyubasha198730@mail.ru (L.V. Kostryukova), burova13@gmail.com (Y.A. Tereshkina), elena.tikhonova_@
mail.ru (E.G. Tikhonova), galina.morozevich@ibmc.msk.ru (G.E. Morozevich), anna2031@rambler.ru (A.D. Plutinskaya), alupatov@mail.ru (A.Yu. Lupatov),
andreimnioi@yandex.ru (A.A. Pankratov).

https://doi.org/10.1016/j.biopha.2020.111154
Received 3 November 2020; Received in revised form 9 December 2020; Accepted 14 December 2020
Available online 25 December 2020
0753-3322/© 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
T.I. Torkhovskaya et al.
Ce6 on healthy tissues. Skin tissue is particularly vulnerable to the
damaging effects of Ce6 due to constant exposure to natural light. These
disadvantages can be overcome by Ce6 administration in the form of
derivatives or as part of various transport systems rather than in its free
(classical) form. In recent years, such systems (liposomes and various
nanoparticles) have been developed in many laboratories [5].
One of them was developed at the Institute of Biomedical Chemistry
(Moscow, Russia). It is an emulsion of ultra-small nanoparticles (less
than 30 nm in size) from vegetable (soy) phosphatidylcholine. When
Ce6 is incorporated into such nanoparticles, it preserves all its spectral
and fluorescent characteristics and the ability to generate ROS. In mice
with a tumor, we revealed a higher accumulation of Ce6 in the tumor
tissue and less in the skin compared to free Ce6 [6].
Recently, we have conducted a number of studies to optimizing this
system using modern approaches to enhance the delivery of transported
drugs to target tissues. This is the attachment of specific cell-penetrating
or/and targeted peptides to the surface of nanoparticles [7]. The vector
effect of the peptides is due to their affinity for a number of receptor
proteins overexpressed on tumor cells [8]. Also, additional attachment
of a cell-penetrating peptide - heptaаrginine, R7 [9], or a peptide with
the NGR sequence (with affinity to aminopeptidase N [10]) to the
phospholipid nanosystem led to increased internalization of Ce6 in the
HерG2 cells. However, we did not evaluate the specific photoinduced
effect of Ce6 in these studies. On the HT-1080 cells, when both peptides
were introduced into the system, we observed an increase in Ce6
accumulation and its photoinduced action compared with the system
without peptides. In the latter case, only the addition of R7 affected the
Ce6 activity. Embedding of NGR enhanced cell accumulation and
internalization but did not increase the antitumor activity of the
photosensitizer, despite the expression of CD13 on these cells [11].
As aminopeptidase N (CD13) is not expressed on a number of tumor
cells, and for the MCF-7 cells that did not respond to the presence of NGR
in the added system [10], the information in this regard is contradictory
[12,13]. Thus, in this work, we aimed at revealing the presence or
absence of CD13 on the MCF-7 and HерG2 cells. Here, we show the
results of the effect of R7 and/or NGR peptide attachment to phospho­
lipid nanoparticles on the addition and internalization of Ce6 embedded
in phospholipid nanoparticles, its photoinduced and dark cytotoxicity in
the experiment on the MCF-7 and HерG2 cells.
2. Materials and methods
2.1. Cell culture
We used cell cultures obtained from the American Type Culture
Collection (ATCC) and maintained in the IBMC Cell Culture Collection –
human hepatocellular carcinoma (HepG2) and human breast adeno­
carcinoma (MCF-7). The cells were cultured according to the recom­
mendations specified in the cell culture certificates, using appropriate
media with the addition of 10 % Fetal Calf Serum (FCS) (PanEco, Russia)
[14]. Cultivation was carried out at 37 ◦ C in a humid atmosphere with
5% CO2 (Binder CO2 incubator, Germany). During passaging, Versen
solution was used to remove cells from the substrate. For this purpose,
the solution was added to the vial for 2− 3 min. We used cell lines from 3
to 10 passages.
2.2. Flow cytometry
Phycoerythrin (PE) conjugated monoclonal antibodies (MoAbs)
against CD13 and isotype control antibodies (BD Bioscience, USA) were
used for immunostaining. 106 cells were incubated with 10 μL of MoAbs
or isotype control antibodies in PBS supplemented with 2% FBS for 30
min at 4 ◦ C. Than, the samples were washed twice with PBS (PanEco,
Russia) and fixed in BD Cytofix™ buffer (BD Bioscience, USA). Fluo­
rescence intensity and light scattering were measured by FACSAriaIII
flow cytometer (BD Bioscience, USA).
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Biomedicine & Pharmacotherapy 134 (2021) 111154
2.3. Preparation of compositions with Ce6 embedded in phospholipid
nanoparticles
To obtain phospholipid compositions with Ce6 and peptides, soy
phosphatidylcholine (Lipoid S100, Lipoid, Germany), di-N-
methylglucamine salt of Ce6 (Ivanovo State University of Chemistry
and Technology, Russia), heptaаrginine R7 (IBMC, Moscow), hexapep­
tide containing the NGR sequence (NH2-)Gly-Asn-Gly-Arg-Gly-Cys
(-COOH) (SintonLab, Saint Petersburg), DSPE–PEG2000–Mal (Avanti,
USA).
Preparation of phospholipid nanoparticles with Ce6 (Nph-Ce6) was
carried out by homogenization of a coarse water emulsion of Ce6 and
phospholipid (at a mass ratio of 1:10) on the Bandelin Sonopuls ultra­
sonic homogenizer (Bandelin, Germany) (KE72 rod, for 6 min at 50 %
power) similar to earlier studies [10,11].
The preparation of a composition with a targeted component (NPh-
Ce6-NGR) was performed similarly [10] using the film method. First, we
obtained the conjugate DSPE-PEG2000-NGR according to the method
described in [10,15], with minor modifications, using the molar ratio of
the main components of DSPE-PEG2000-Mal : NGR 4.3:1. Then, phos­
pholipid Lipoid S100 was dissolved in 96 % ethanol (Medkhimprom,
Russia) in a weight ratio of 125:1; separately, the conjugate
DSPE-PEG2000-NGR was dissolved in 96 % ethanol in the ratio of 25:1
(weight ratio). The resulting alcohol solutions were mixed in the ratio of
Lipoid: conjugate 10:1 (by volume). Alcohol was removed on a rotary
evaporator Heidolph Laborota 4003 (Heidolph, Germany) until a dry
film was obtained. The resulting film was rehydrated with an aqueous
solution of Ce6 (based on the mass ratio of Ce6: Lipoid 1:10). The coarse
emulsion was processed on an ultrasonic disintegrator similar to
NPh-Ce6, as described above.
To obtain compositions with a cell-penetrating peptide (NPh-Ce6-
R7) or containing both peptides (NPh-Ce6-NGR-R7), R7 peptide was
added to the previously obtained NPh-Ce6 (or NPh-Ce6-NGR, respec­
tively) in a ratio of 6:1 to Ce6 and shaken vigorously on the IKA MS 3
basic shaker (IKA, USA). All ultrathin emulsions obtained were passed
through a 220 nm filter (Merck Millipore, USA). In all samples, the
particle size, ζ-potential (Malvern, UK) and the percentage of Ce6
incorporation into phospholipid nanoparticles were determined using
the Agilent 1100 series HPLC chromatography system (Agilent Tech­
nologies, USA).
2.4. Evaluation of cell binding and Ce6 penetration ability in the studied
compositions
The HepG2 and MCF-7 cells were dispersed into 6-well culture plates
at a concentration of 106 cells per well and incubated for 24 h at 37 ◦ C.
Then, the samples obtained were added to the cells at a concentration of
25 μg/mL (for Ce6) and incubated for 4 h at 37 ◦ C in a CO2 incubator
(Sanyo, Japan) and 4 ◦ C.
For all three compositions of Ce6 in phospholipid nanoparticles
equipped with specific peptides: cell-penetrating, targeted or their
combination (respectively, NPh-Ce6-R7, NPh-Ce6-NGR, and NPh-Ce6-
NGR-R7), after incubation with both types of cells, accumulation of
Ce6 in the cells, its attachment to the cell surface, and penetration into
the cells (internalization) were studied. The initial Ce6 nano­
phospholipid system without peptides (NPh-Ce6) was used as compar­
ison objects.
After incubation, the medium with the studied compositions was
removed, the cells were washed twice with PBS. Determination of the
Ce6 content in the cells was performed after its extraction with 0.1 %
formic acid (Sigma, USA) in methanol (Fisher Scientific, UK) (1 mL per
well), as described earlier [6,9]. The extracts were transferred to
Eppendorf tubes and centrifuged at 10000 rpm for 10 min (at 4 ◦ C)
(Eppendorf 5810R, FA rotor-45-30-11, Eppendorf, Germany), then
analyzed by HPLC on Agilent 1200 Series with an Eclipse XDB-C18
column (Agilent Technologies, USA) and 6130 Quadrupole LC/MS
T.I. Torkhovskaya et al. Biomedicine & Pharmacotherapy 134 (2021) 111154

mass spectrometry detector (Agilent Technologies, USA). The Ce6 con­

tent in the cells was normalized to the level of protein (mg) determined
by the Lowry method. The total Ce6 penetration (internalization) into
the cells was calculated from the difference in its content at 37 ◦ C (total
accumulation in cells) and at 4 ◦ C [16].

2.5. Assessment of photoinduced activity

The cells were dispersed into sterile 96-well culture plates at a con­
centration of 105 cells per well and incubated at 37 ◦ C in an atmosphere
of 5% CO2 for 24− 26 hours. Next, the compositions compared (NPh-
Ce6, NPh-Ce6-R7, NPh-Ce6-NGR, or NPh-Ce6-NGR-R7) were added to
the plates in the Ce6 concentrations from 0.016 μg/mL to 20 μg/mL
(samples were diluted in step 5 with a culture medium without serum).
The cells were incubated with the samples for 24 h at 37 ◦ C in an at­
mosphere of 5% CO2. After incubation and washing with the PBS buffer,
the cells were exposed to light (the source of irradiation was a halogen
lamp (Russia) with a wide-band filter KC-13 (λ ≥ 640 nm), with a power
of 500 W). The light dose was 10 J/cm2. After irradiation the cells were
left under standard conditions (37 ◦ C in an atmosphere of 5% CO2) for
24− 28 hours; the survival of tumor cells was evaluated using the MTT
test (Sigma, USA). The absorption of formazan added during this test
was recorded at 550 nm (Multiscan FC, Thermo Spectronic, USA). The
percentage of live cells in the medium was determined by the change in
absorption when normalized to the untreated control. The criterion for
evaluating specific activity was the IC50 value (the concentration of Ce6,
which causes 50 % of tumor cell death) [14].

2.6. Assessment of non-specific cytotoxicity without light exposure

To determine the non-specific cytotoxic effect of the studied Ce6

samples in the delivery systems, the HepG2 and MCF-7 cells were sown
in a 96-well plate (105 cells/well) and cultured under standard condi­
tions of 37 ◦ C in an atmosphere of 5% CO2 for 24− 26 hours. Further, all
stages were performed similarly to the assessment of photoinduced ac­
tivity, excluding the irradiation stage. Cell viability was evaluated using
the MTT test. The percentage of live cells was estimated by fluorescence
as a percentage of that in the system without Ce6 added.

2.7. Statistical analysis

Statistical significance of the data was analysed through independent

sample t test by SPSS Statistics R24.0 software (IBM). The figures and the
tables show the data as mean ± standard error of the mean. Probability
value of <0.05 was considered statistically significant (*P < 0.05).
3. Results
3.1. Expression of aminopeptidase N on HepG2 and MCF-7 cells
The protein aminopeptidase N (CD13) was first identified and
characterized in a number of studies on tumor vessels as a target for the
NGR containing peptides [17]. It is a transmembrane glycoprotein that
catalyzes the hydrolysis of the N-terminal peptide bond of proteins and
peptides [18]. Its increased expression was subsequently shown on
tumor cells, which aroused interest in this protein in terms of antitumor
drug delivery using the NGR peptide as a vector [8,10,17]. As our
research was aimed at developing an optimal delivery system for an
effective anticancer drug, chlorin e6, it was interesting to find out
whether the NGR peptide could be used for this purpose. Due to its
interaction with the CD13 protein expressed on cells, it is possible to
improve the Ce6 delivery to tumor cells.
We used flow cytometry to assess the difference in CD13 expression
between two cell lines that we planned to use in future experiments on
NGR peptide dependent delivery. The flow cytometry data demon­
strated a rather high level of expression of CD13 on HepG2 cell surface
Fig. 1. Expression of CD13 on the HepG2 (a) and MCF-7(b) cell lines (flow
cytometry data). The cells were stained with PE-conjugated monoclonal anti­
bodies against CD13 (broken line) or isotype control antibodies (solid line). X-
axis (PE-A: CD13) - fluorescence intensity, relative units, Y-axis (Count) - cell
quantity in the flow.
(Fig. 1a). In contrast, no expression of CD13 was detected on MCF-7 cells
(Fig. 1b), which implies that this pair of cell lines can be used in
comparative experiments. It should be noted that the expression of the
protein aminopeptidase N (CD13) in HepG2 cells was also observed by
other authors [19,20].
3.2. Characterization of phospholipids nanoparticles with embedded Ce6
Then, these two cell lines with different CD13 expression were
treated with phospholipid compositions of Ce6 with the specific peptides
attached. The main characteristics of these compositions are shown in
Table 1.
As it is seen, all four compositions constituted ultrasmall nano­
particles with a diameter up to 33 nm and high Ce6 inclusion. ζ-potential
was negative because of Ce6 carboxyl groups. And, it was neutralized by
arginine positive charge in two compositions with R7.

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T.I. Torkhovskaya et al. Biomedicine & Pharmacotherapy 134 (2021) 111154

Table 1 compared to the phospholipid nanoform NPh-Ce6. The maximum

Physical and chemical characteristics of Ce6 compositions. impact on Ce6 accumulation and especially internalization (more than 2
Properties NPh- NPh-Ce6- NPh-Ce6- NPh-Ce6 times compared to the initial nanoparticles, without peptides) was
Ce6-R7 NGR NGR-R7 exerted by the combination of two peptides (right columns). Cell
Particle size, D nm 29.5 ± 25.3 ± 30.8 ± 2.2 18.1 ± internalization of Ce6 reached an average of 0.16 μg/protein mg
Fraction of particles of a 2.2 5.05 (77.0 ± 4.9 comparing to 0.7 μg/protein mg for the system without peptides. The
specified size,% (85.2 ± (90.1 ± 9.6) (96.4 ± effect of R7 and NGR separately (for NPh-Ce6-R7 and NPh-Ce6-NGR
8.5) 6.3) 3.2) systems) was almost identical. The attachment of Ce6 to the cell sur­
ζ- potential, mV 4.97 ± − 20.0 ± 6.21 ± 2.0 − 23.3 ±
3.4 4.3 6.6
face also increased equally for all three compositions - 2–3 times
Level of Ce6 inclusion in 99.9 ± 98.9 ± 0.7 99.9 ± 96.5 ± compared to the system without peptides. Each of the peptides used
nanoparticles, % 0.2 0.14 7.3 separately approximately doubled the attachment.
When evaluating the accumulation of Ce6 on the MCF-7 cell line,
there was no noticeable effect for any of the systems, which indicated
3.3. Accumulation and internalization of Ce6 in HepG2 and MCF-7 cells
the lack of influence of specific peptides. There was only a slight (by
20–30 %, compared to 2–3 times for HepG2) increase in the attachment
As seen in Fig. 2a, in the HepG2 cells, the total accumulation (at 37
of Ce6 to the cell surface for compositions with R7 (alone or in combi­
◦
C) of Ce6 for all compositions with peptide fragments was higher
nation with NGR). However, such an increase did not affect its

Fig. 2. Effect of attaching cell-penetrating R7 and/or specific NGR-motif peptides to the transport system on the accumulation of Ce6 in the HepG2 (a) and MCF-7 (b)
cell cultures after 4 h of incubation.

4
T.I. Torkhovskaya et al.
internalization in the cells – it did not change compared with the initial
system without peptides.
3.4. Photoinduced activity of chlorin e6 in the phospholipid nanoparticles
with peptides
When using the initial Ce6 nanoparticles without adding peptides,
the IC50 values were close to one another for both cell lines, on average
2.6 μg/mL (the difference between them was not statistically significant)
(Table 2). A considerable decrease in the IC50 value, indicating
increasing specific light-induced cytotoxic effect of the photosensitizer,
was observed for the two systems containing one penetrating peptide
(R7) or in combination with NGR. The most pronounced decrease in the
IC50 value (3 times, up to 0.8 μg/mL) was observed for the HepG2 cells.
The effect was somewhat less noticeable on the MCF-7 cells, but
compared to the system without peptides, it was more than 2 times
higher (IC50 1.3–1.4 μg/mL compared to 3.0 ± 0.9 μg/mL). However,
the addition of NGR to the system with R7 did not increase the effec­
tiveness of tumor cells in any of the cultures. The expected activating
effect of the NGR vector peptide was not detected either. In both cell
cultures, the IC50 value for the NGR peptide composition was compa­
rable to the phospholipid Ce6 nanoform (NPh-Ce6), despite its stimu­
lating effect on the cell accumulation and internalization in the HepG2
cells shown above (Fig. 2).
3.5. Cytotoxic effect of Ce6 on cells in different delivery systems without
light irradiation
Both the photoinduced activity of photosensitizers and their non-
specific effect on cells without light irradiation should be taken into
account when evaluating the possibility of their use in PDT in the de­
livery systems [21]. This is important in terms of their possible pene­
tration into healthy organs not exposed to radiation during PDT. For a
medicinal photosensitizing drug, this effect should be minimal, signifi­
cantly less than that caused by light. We have evaluated all nano­
phospholipid compositions of Ce6 on the HepG2 and MCF-7 cells. Fig. 3
shows the survival of cells remained in the medium after incubation with
the studied Ce6 compositions, without light irradiation, as a percentage
of the level without adding Ce6 (according to the MTT test). To show a
significant predominance of photoinduced activity over nonspecific
dark activity of the same systems, the measurement was performed at
concentrations of Ce6 both close to IC50 (1.25 and 5 μg/mL) and many
times higher, up to 20 μg/mL.
As seen from Fig. 3, Ce6 included in phospholipid nanoparticles at
the first two concentrations (1.25 and 5 μg/mL) did not show dark
cytotoxicity on both types of cells. At a concentration of 20 μg/mL, a
small (about 6–8 %) decrease in the number of living cells was observed
on the MCF-7 and HepG2 cells, which slightly reduced (statistically
unreliable) in the variants with peptides. In general, the cytostatic effect
of all the phospholipid compositions studied was practically absent.
Thus, the addition of selected peptides to the phospholipid transport
nanosystem containing Ce6 does not enhance its «dark» toxicity, and it
remains significantly lower than the specific antitumor photodynamic
activity.
Table 2
Photoinduced activity of Ce6 in phospholipid nanoparticles after the addition of
specific targeted (NGR) or/and penetrating (R7) peptides.
IC50 value (μg/mL) for cell cultures
Ce6 composition
HepG2 MCF-7
NPh-Ce6 2.3 ± 0.2 3.0 ± 0.9
NPh-Ce6 -R7 0.8 ± 0.2 1.4 ± 0.4
NPh-Ce6 -NGR 1.9 ± 0.3 3.8 ± 0.2
NPh-Ce6 -NGR–R7 0.8 ± 0.2 1.3 ± 0.3
5
Biomedicine & Pharmacotherapy 134 (2021) 111154
4. Discussion
Among various pharmacological targets of anticancer therapy, the
protein aminopeptidase N (APN) occupies a particular place. It is
considered a possible dual-target in antitumor therapy, both in terms of
its inhibition to activate apoptosis diminished by this protein, and as a
target for targeted drug delivery and drug systems [22,23]. Thus,
attachment of the peptides with amino acid sequence NGR affine to
aminopeptidase N to these systems may become an effective method [8,
24–26].
The studies are being carried out in this area, and several selected
medicinal compositions are undergoing preclinical testing. At the same
time, the approach of using APN as a specific target for drug delivery
into a tumor is not yet considered sufficiently established. This protein,
as noted by Wickström et al. [22], has a number of insufficiently clari­
fied («moon-lighting») functions. Its tumor expression is not specific; it
does not always correlate with the stage and severity of the disease and
is not detected, for example, in some types of breast cancer [27].
Therefore, further research is considered necessary to evaluate the
possibility of the future therapeutic use of this protein expression [22].
The expression of CD13 on the HepG2 cells recorded in our study using
flow cytometry coincides with the data reported by other authors [19].
The other selected cell type, MCF-7, is noted in most studies as
CD13-negative (it does not contain this protein) [12]. Our data
confirmed the absence of CD13 expression on these cells. The obtained
results were the basis for a comparative study focusing on these two
types of cells, which was aimed at revealing the effect of the addition of
NGR peptide with an affinity for CD13 to phospholipid nanoparticles
containing the photosensitizer Ce6. The peptide was also attached in
combination with the cell-penetrating peptide heptaarginine R7.
The attachment of an NGR-containing peptide to the Ce6 phospho­
lipid nanosystem increased the accumulation of this photosensitizer
only on the HepG2 cells expressing aminopeptidase N and did not affect
MCF-7. For the latter, only a small impact of the cell-penetrating peptide
R7 was observed, which slightly enhanced Cе6 attachment to the cell
surface. This may be due to the ability of phospholipid nanoparticles,
remaining only in the area of the membrane, to prevent its further
penetration. A similar effect of R7 was shown for the HepG2 cells, where
both peptides (separately) equally increased the attachment and inter­
nalization of Ce6. The maximum internalization was observed for their
combination – in this case, R7 somehow «helped» Ce6 attached via APN
to enter the cell. For MCF-7, however, the situation was different – due
to the absence of CD13 and, as a consequence, the additionally attached
photosensitizer. In general, the attachment of both peptides (separately
or in combination) contributed to an increase in the accumulation of Ce6
introduced as part of phospholipid nanoparticles, compared with the
action of the same nanoparticles without peptides.
At the same time, despite the difference in the character of cellular
accumulation, the degree of influence of the peptides on the specific
photoinduced activity of Ce6 in phospholipid nanoparticles was almost
the same for both types of cells. For the two systems containing the cell-
penetrating peptide R7 (regardless of the NGR presence), the IC50 values
of Ce6 decreased by 2.5–3 times compared to the phospholipid system
without peptides, indicating higher Ce6 activity. The use of such pep­
tides, most often containing arginine, is considered as an effective
approach to increasing drug penetration into the cell, by conjugating
them with the drug or embedding in transport nanoparticles.
The precise mechanism of cellular internalization of Arg containing
peptides is still discussed [28]. Some authors suggest that short
cell-penetrating peptides with attached groups can be translocated
across the membrane by an energy-independent and non-receptor
mechanism. This is indicated by the preservation of their ability to
internalize in the presence of endocytosis inhibitors, as well as with a
decrease in temperature [29,30]. At the same time, there is evidence for
an endocytosis mechanism, especially for the peptides most rich in
arginine [31]. The guanidine group of Arg is believed to be the
T.I. Torkhovskaya et al.
Fig. 3. Cytotoxicity (without light exposure) of Ce6 on the HepG2 (a) and MCF-7 (b) cell lines measured in various delivery systems at its different concentrations in
the medium.
determining factor for internalization [32]. In our experiments, R7
increased only attachment of Ce6 to the cell surface, and its internali­
zation and translocation through the membrane were not observed.
However, it was the addition of the R7 peptide that had the most pro­
nounced stimulating effect on the activity of Ce6 after light irradiation.
This can be explained based on the mechanism of oligoarginines pene­
tration into the cell proposed in [33]. According to the authors, the
guanidine groups of arginine induce the micro-damages in a number of
membrane domains, and thus the peptides enter the cell. It can be
assumed that the size of the micro-damage is insufficient for the passage
of phospholipid nanoparticle, and it seems to «get stuck» and retain in
the membrane. When irradiated with light, the Ce6 attached undergoes
its typical photodynamic reaction with the generation of ROS. The
irradiation products have a cytotoxic effect. It can be realized both after
ROS penetration into cells and by the action of the oxidation products of
unsaturated fatty acid of phospholipids. The objects of oxidation can be
the phospholipids both of nanoparticles themselves and of the cell
membrane. These effects lead to the cell death observed.
In the HepG2 cells, with NGR expression, we observed a slight in­
crease in the internalization of Ce6 embedded in nanoparticles with
NGR. However, there was no effect of NGR on the Ce6 impact. Thus, in
these cells, the more significant impact is caused by the photosensitizer
attached, i.e., the products of its photoinduced reactions, rather than by
that penetrated into cell. The effect of NGR attachment to the Ce6
phospholipid system can be clarified in more detail in experiments in
vivo, where the tumor specificity of this peptide for APN should appear,
6
Biomedicine & Pharmacotherapy 134 (2021) 111154
as well as a decrease in its relative intake to normal tissues and,
consequently, reduction of side effects. The data on the absence of
cytostatic action of all the phospholipid compositions studied without
light irradiation indicate the absence of the risks of increased side effects
when peptides are added to nanoparticles, as well as for nanoparticles
themselves. The slight «dark» effect characteristic of Ce6 is much
weaker than the photodynamic activity enhanced in the systems
developed due to the addition of a cell-penetrating peptide.
Thus, the inclusion of Ce6 photosensitizer in phospholipid nano­
particles equipped with specific peptides can be considered as a prom­
ising way to increase the efficiency of PDT. Further research using
animal tumor models will provide information on the possible efficacy
of the attachment of targeted NGR peptide to phospholipid nano­
particles for their targeted delivery to the cells expressing aminopepti­
dase N.
Funding
The work was performed in the framework of the Program for Basic
Research of State Academies of Science.
Declaration of Competing Interest
The authors report no declarations of interest.
T.I. Torkhovskaya et al. Biomedicine & Pharmacotherapy 134 (2021) 111154

References [17] R. Pasqualini, E. Koivunen, R. Kain, J. Lahdenranta, M. Sakamoto, A. Stryhn, R.

A. Ashmun, L.H. Shapiro, W. Arap, E. Ruoslahti, Aminopeptidase is a receptor for
tumor-homing peptides and a target for inhibiting angiogenesis, Cancer Res. 60 (3)
[1] B.M. Luby, C.D. Walsh, G. Zheng, Advanced photosensitizer activation strategies
(2000) 722–727.
for smarter photodynamic therapy beacons, Angew. Chem. Int. Ed. Engl. 58 (9)
[18] X. Wang, B. Wang, Q. Zhang, Anti-tumor targeted drug delivery systems mediated
(2019) 2558–2569, https://doi.org/10.1002/anie.201805246.
by aminopeptidase N (CD13), Acta Pharm. Sinica B 1 (2) (2011) 80–83.
[2] R.R. Allison, K. Moghissi, Photodynamic therapy (PDT): PDT mechanisms, Clin.
[19] S. Saida, K. Watanabe, I. Kato, H. Fujino, K. Umeda, S. Okamoto, S. Uemoto,
Endosc. 46 (1) (2013) 24–29.
T. Hishiki, H. Yoshida, S. Tanaka, S. Adachi, A. Niwa, T. Nakahata, T. Heike,
[3] A. Juarranz, P. Jaen, F. Sanz-Rodriguez, S. Cuevas, J. Gonzalez, Photodynamic
Prognostic significance of aminopeptidase-N (CD13) in hepatoblastoma, Pediatr.
therapy of cancer. Basic principles and applications, Clin. Transl. Oncol. 10 (2008)
Int. 57 (4) (2015) 558–566, https://doi.org/10.1111/ped.12597.
148–154.
[20] H. Li, Y. Li, Q. Yao, J. Fan, W. Sun, S. Long, K. Shao, J. Du, J. Wang, X. Peng, In situ
[4] A. Juzeniene, Chlorin e6-based photosensitizers for photodynamic therapy and
imaging of aminopeptidase N activity in hepatocellular carcinoma: a migration
photodiagnosis, Photodiagnosis Photodyn. Ther. 6 (2) (2009) 94–96.
model for tumour using an activatable two-photon NIR fluorescent probe, Chem.
[5] B. Chen, B.W. Pogue, T. Hasan, Liposomal delivery of photosensitising agents,
Sci. 10 (6) (2018) 1619–1625, https://doi.org/10.1039/c8sc04685a.
Expert Opin. Drug Deliv. 2 (2005) 477–487.
[21] Y. Baglo, A.J. Sorrin, B.J. Liang, H.C. Huang, Harnessing the potential synergistic
[6] L.V. Kostryukova, V.N. Prozorovskiy, N.V. Medvedeva, O.M. Ipatova, Comparison
interplay between photosensitizer dark toxicity and chemotherapy, Photochem.
of a new nanoform of the photosensitizer chlorin e6, based on plant phospholipids,
Photobiol. 96 (3) (2020) 636–645, https://doi.org/10.1111/php.13196.
with its free form, FEBS Open Bio 8 (2) (2018) 201–210.
[22] M. Wickström, R. Larsson, P. Nygren, J. Gullbo, Aminopeptidase N (CD13) as a
[7] G.M.F. Calixto, J. Bernegossi, L.M. de Freitas, C.R. Fontana, M. Chorilli,
target for cancer chemotherapy, Cancer Sci. 102 (3) (2011) 501–508.
Nanotechnology-based drug delivery systems for photodynamic therapy of cancer:
[23] S.A. Amin, N. Adhikari, T. Jha, Design of aminopeptidase N inhibitors as anti-
a review, Molecules 21 (2016) 342.
cancer agents, J. Med. Chem. 61 (15) (2018) 6468–6490.
[8] V.N. Prozorovskiy, T.I. Torkhovskaya, L.V. Kostryukova, O.M. Ipatova, Specific
[24] C.L. Schreiber, B.D. Smith, Molecular imaging of aminopeptidase N in cancer and
peptides usage for targeted delivery of nanoparticles with antitumor drugs, Russ. J.
angiogenesis, Contrast Media Mol. Imaging (2018), 5315172, https://doi.org/
Biopharm. 10 (4) (2018) 3–18.
10.1155/2018/5315172.
[9] L.V. Kostryukova, E.I. Korotkevich, G.E. Morozevich, E.F. Kolesanova, M.
[25] A. Corti, A.M. Gasparri, A. Sacchi, B. Colombo, M. Monieri, E. Rrapaj, A.J.
V. Mel`nikova, Y.V. Filatova, T.I. Torkhovskaya, V.N. Prozorovskii, E.
M. Ferreri, F. Curnis, NGR-TNF engineering with an n-terminal serine reduces
G. Tikhonova, O.M. Ipatova, Effect of cell-penetrating arginine peptide on
degradation and post-translational modifications and improves its tumor-targeting
interaction of photosensitizer chlorine e6 incorporated into phospholipid
activity, Mol. Pharm. 17 (10) (2020) 3813–3824, https://doi.org/10.1021/acs.
nanoparticles with tumor cells, Bull. Exp. Biol. Med. 167 (3) (2019) 347–350.
molpharmaceut.0c00579.
[10] V.N. Prozorovskiy, L.V. Kostryukova, E.I. Korotkevich, T.I. Torkhovskaya, G.
[26] Y. Huang, Q. Cheng, X. Jin, J.L. Ji, S. Guo, S. Zheng, X. Wang, H. Cao, S. Gao, X.
E. Morozevich, E.G. Tikhonova, O.M. Ipatova, Photosensitizer chlorine e6
J. Liang, Q. Du, Z. Liang, Systemic and tumor-targeted delivery of siRNA by cyclic
internalization into tumor cells in phospholipid nanoparticles conjugated with
NGR and isoDGR motif-containing peptides, Biomater. Sci. 4 (3) (2016) 494–510,
peptide containing the NGR sequence, Biomed. Chem. Res. Methods 1 (4) (2018),
https://doi.org/10.1039/c5bm00429b.
e00063, https://doi.org/10.18097/bmcrm00063.
[27] S. Elgun, M. Karaayvaz, I. Durak, Alanine aminopeptidase activities in cancerous
[11] L.V. Kostryukova, A.D. Plyutinskaya, A.A. Pankratov, E.I. Korotkevich, V.
and non-cancerous human breast tissues, Eur. J. Clin. Chem. Clin. Biochem. 35
N. Prozorovskiy, E.G. Tikhonova, T.I. Torkhovskaya, Yu.A. Teryoshkina, Chlorine
(1997) 249.
e6 in phospholipid nanoparticles with specific targeting and penetrating peptides
[28] F. Heitz, M.C. Morris, G. Divita, Twenty years of cell-penetrating peptides: from
as prospective composition for photodynamic therapy of malignant neoplasms,
molecular mechanisms to therapeutics, Br. J. Pharmacol. 157 (2009) 195–206.
Biochem. Moscow Suppl. Ser. B 14b (2020) 174–179, https://doi.org/10.1134/
[29] S. Dissanayake, W.A. Denny, S. Gamage, V. Sarojini, Recent developments in
S1990750820020080.
anticancer drug delivery using cell penetrating and tumor targeting peptides,
[12] L. Hou, X. Zhao, P. Wang, Q. Ning, M. Meng, C. Liu, Antitumor activity of
J. Control. Release 250 (2017) 62–67.
antimicrobial peptides containing CisoDGRC in CD13 negative breast cancer cells,
[30] P.A. Wender, D.J. Mitchell, K. Pattabiraman, E.T. Pelkey, L. Steinman, J.
PLoS One 8 (1) (2013), e53491, https://doi.org/10.1371/journal.pone.0053491.
B. Rothbard, The design, synthesis, and evaluation of molecules that enable or
[13] J. Dixon, L. Kaklamanis, H. Turley, I.D. Hickson, R.D. Leek, A.L. Harris, K.C. Gatter,
enhance cellular uptake: peptoid molecular transporters, Proc. Natl. Acad. Sci. U. S.
Expression of aminopeptidase-N (CD 13) in normal tissues and malignant
A. 97 (2000) 13003–13008, https://doi.org/10.1073/pnas.97.24.13003.
neoplasms of epithelial and lymphoid origin, J. Clin. Pathol. 47 (1) (1994) 43–47,
[31] S. Futaki, I. Nakase, A. Tadokoro, T. Takeuchi, A. Jones, Arginine-rich peptides and
https://doi.org/10.1136/jcp.47.1.43.
their internalization mechanisms, Biochem. Soc. Trans. 35 (2007) 784–787.
[14] R.I. Yakubovskaya, N.I. Kazachkina, T.A. Karmakova, N.B. Morozova, A.
[32] J.B. Rothbard, T.C. Jessop, R.S. Lewis, B.A. Murray, P.A. Wender, Role of
A. Pankratov, A.D. Plyutinskaya, A.V. Feofanov, V.I. Chissov, A.I. Zebrev, A.
membrane potential and hydrogen bonding in the mechanism of translocation of
V. Tikhomirova, in: A.N. Mironov (Ed.), Guide to Preclinical Preclinical Drug
Guanidinium-rich peptides into cells, J. Am. Chem. Soc. 126 (2004) 9506–9507.
Research, Grif and co., Moscow, 2012, pp. 655–669.
[33] T. Murayama, T. Masuda, S. Afonin, K. Kawano, T. Takatani-Nakase, H. Ida,
[15] Y. Yang, Y. Yang, X. Xie, X. Cai, H. Zhang, W. Gong, Z. Wang, X. Mei, PEGylated
Y. Takahashi, T. Fukuma, A.S. Ulrich, S. Futaki, Loosening of lipid packing
liposomes with NGR ligand and heat-activable cell-penetrating peptide-
promotes oligoarginine entry into cells, Angew. Chem. Int. Ed. Engl. 56 (26) (2017)
doxorubicin conjugate for tumor-specific therapy, Biomaterials 35 (14) (2014)
7644–7647.
4368–4381, https://doi.org/10.1016/j.biomaterials.2014.01.076.
[16] K. Sheldon, D. Liu, J. Ferguson, J. Gariépy, Loligomers: design of de novo peptide-
based intracellular vehicles, Proc. Natl. Acad. Sci. U. S. A. 92 (6) (1995)
2056–2060.