Plasmin-Binding Tripeptide-Decorated Liposomes Loading Pyrazolo[3,4- d]pyrimidines for Targeting Hepatocellular Carcinoma

Article abstract of DOI:10.1021/acsmedchemlett.8b00062

Hepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo[3,4-d]pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo[3,4-d]pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. In vitro HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds.

Full text of DOI:10.1021/acsmedchemlett.8b00062

Letter
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Plasmin-Binding Tripeptide-Decorated Liposomes Loading
Pyrazolo[3,4‑d]pyrimidines for Targeting Hepatocellular Carcinoma
Pierpaolo Calandro,†,‡,○ Giulia Iovenitti,†,○ Claudio Zamperini,†,§ Francesca Candita,† Elena Dreassi,†
Mario Chiariello,‡ Adriano Angelucci,∥ Silvia Schenone,⊥ Maurizio Botta,*,†,§,# and Arianna Mancini†,∇
†Dipartimento Biotecnologie, Chimica e Farmacia, Universita
̀
degli Studi di Siena, Via A. Moro 2, I-53100 Siena, Italy
‡Consiglio Nazionale delle Ricerche, Istituto di Fisiologia Clinica and Istituto per lo Studio, la Prevenzione e la Rete Oncologica
(ISPRO), Core Research Laboratory, Via Fiorentina 1, 53100 Siena, Italy
§Lead Discovery Siena S.r.l., Via Vittorio Alfieri 31, 53019 Castelnuovo Berardenga, Siena, Italy
∥Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Universita
̀
degli Studi dell’Aquila, Via Vetoio, 67100, Coppito,
L’Aquila, Italy
⊥Dipartimento di Farmacia, Universita
̀
degli Studi di Genova, Viale Benedetto XV, 3, 16132 Genova, Italy
#Biotechnology College of Science and Technology, Temple University, Biolife Science Building, Suite 333, 1900 N 12th Street,
Philadelphia, Pennsylvania 19122, United States
∇Dipartimento di Farmacia, Universita
̀
di Pisa, Via Bonanno Pisano 6, 56126 Pisa, Italy
S
* Supporting Information
ABSTRACT: Hepatocellular carcinoma (HCC) is one of the most fatal
cancer types worldwide. HCC cells were proved to overexpress c-Src and
Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is
crucial for the development and progression of the tumor. Pyrazolo[3,4-
d]pyrimidine derivatives are a class of tyrosine kinase inhibitors that have
shown good activity against HepG2. HCC cells were also proved to
overexpress plasmin, which is localized on the cell surface bound to its
receptors. In this study, a tripeptide with sequence D-Ala-Phe-Lys, which
binds a specific reactive site of plasmin, was synthesized and characterized.
This tripeptide was used to decorate liposomes encapsulating three selected
pyrazolo[3,4-d]pyrimidines. Liposomes bearing tripeptide have been
characterized, not showing remarkable differences with respect to the
corresponding tripeptide-free liposomes. In vitro HepG2 cell uptake profiles
and cytotoxicities showed that the presence of the tripeptide on the
liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three
tested compounds.
KEYWORDS: Hepatocellular carcinoma, active-targeted liposomes, pyrazolo[3,4-d]pyrimidines, plasmin
be used as antineoplastic agents for HCC.7 Pyrazolo[3,4-
epatocellular carcinoma (HCC) is the most common
Hhistological subtype among primary liver malignancies and
d]pyrimidine derivatives are a class of small molecules ATP-
competitive c-Src inhibitors, which have been developed,
one of the most fatal cancer types in the world.1 For intermediate
synthesized, and extensively studied in our laboratories. They
were found to induce apoptosis and proliferation impairment in
several solid cancer cell lines.8−11 Furthermore, one of these
molecules, named SI113, was identified as a potent inhibitor of
both c-Src and Sgk1, a Ser/Thr kinase that has recently gained
attention as molecular target in oncology for its regulating role in
cell survival, proliferation, and differentiation.12,13 In HCC, Sgk1
expression is particularly relevant, and its inhibition by SI113 in
HepG2 and HUH-7 cell lines resulted in a dramatic increase in
and advanced stage (70% of HCC patients), there are palliative
treatments including transarterial chemoembolization (TACE).
Sorafenib, an oral multitargeted kinase inhibitor, has been
approved for the treatment of HCC.2 Despite the available
treatments, the mean survival rate of patients with advanced stage
HCC is less than 1 year.3 As in many other cancer types,4 both
moderately and well differentiated HCC cells were proved to
overexpress c-Src family proteins,5,6 a class of nonreceptor
tyrosine kinases involved in cascades of signaling pathways
essential for cell proliferation, motility, and survival. c-Src
hyperactivation plays a central role in HCC progression and
may be partly responsible for the malignant transformation of
hepatocytes.6 Accordingly, c-Src inhibitors have the potential to
Received: February 8, 2018
Accepted: May 7, 2018
Published: May 7, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00062
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Figure 1. Molecular structures of compounds SI113, S29, and SI163, encapsulated into liposomes and TRP (plasmin-binding tripeptide-linker), used to
decorate liposomal surface.
Table 1. Compounds S29, SI113, SI163: activity against Src and Sgk1, ADME Properties, and IC50 against HepG2 Cells
a
b
c
d
e
compd
Src (Ki, μM)
Sgk1 (residual activity %)
aq solub (μg mL−1
)
Papp (10−6 cm/sec 50% DMSO)
met stab %
IC50 (μM)
S29
0.8
<0.04
0.4
16.6
2.6
94.0
1.6 1.1
12.5 3.4
3.2 2.3
SI113
SI163
2.4
0
≥95.0
93.6
0.0093
0.098
10.0
a
b
c
All data were previously reported.11,36 Residual activity of Src kinase after exposure to 12.5 μM SI113 for 30 min.13 Aqueous solubility was
d
e
determined by means of the LC-UV method. Apparent permeability was determined with PAMPA assay. The human liver microsomal stability is
expressed as percentage of unmodified parent drug.
apoptosis and altered cell cycle. In human hepatocarcinoma cells
xenografted immunodeficient mice, SI113 arrested cancer
growth, inducing high levels of necrosis in tumor tissues.14
However, the main weakness of these molecules is their low
aqueous solubility, which could give rise to low bioavailability
and low target specificity, affecting their pharmacologic activity.
To overcome this issue, liposomal technology has recently been
explored: four pyrazolo[3,4-d]pyrimidine derivatives with
antineuroblastoma activity have been encapsulated in stealth
liposomes and tested in vivo with an improvement in
pharmacokinetic properties and in vitro with retention of
cytotoxic efficacy with respect to free compounds in human
neuroblastoma cell line.15 Liposomes have been extensively
investigated and used as pharmaceutical carriers for antineo-
plastic drugs for their many favorable physicochemical and
pharmacological features and their versatility.16 Ligand-con-
jugated liposomes for active targeting offer the prospect of
improving the effectiveness and minimizing the off-target effects
of anticancer therapy.17 Active delivery of liposomes may provide
increased accumulation at the target tissue, enhanced cellular
internalization, and triggered release of therapeutic payloads,
exploiting pathological peculiarities in the tumor’s microenviron-
ment.18
It is widely acknowledged that the broad-spectrum serine
protease plasmin (PM) and its activation system have a primary
role in cancer metastasis.19 PM is partly responsible, directly or
by activating several pro-metalloproteases (MMPs), for the
degradation of extra-cellular matrix (ECM), disaggregation of
basement lamina, and catabolism of adhesion molecules,
promoting cancer cells invasion. PM is mostly found in its cell
surface-associated form, bound to its receptors (plasminogen
receptors, PG-R),20,21 which are expressed by a wide variety of
cells with high density (>107 binding sites for plasminogen on
some cells),22 especially carcinoma cells.23 This allows for
localized and much greater proteolytic activity and protection
from inactivation (active PM occurs in the blood circulation in
very low concentrations because it is rapidly inhibited by α-
antiplasmin). Some of PG-R are overexpressed on carcinoma
cells, among which are α-enolase (ENO-1),24,25 cytokereatin 8,26
annexin A2,27 amphoterin.28 ENO-1, in particular, was found to
be overexpressed in more than 20 types of human cancer.29 The
imbalance between plasminogen activators (PAs) and PM in
tumor microenvironment is responsible for the excessive
production of PM. Increased expression level of plasminogen
activators has been recognized in many types of malignant
tumors,30−32 including HCC.33
The first part of this work describes the synthesis and the
characterization of a specific tripeptide with sequence D-Ala-Phe-
Lys, which binds a specific reactive site of plasmin.34 The second
part focuses on the functionalization of the outer membrane of
pyrazolo[3,4-d]pyrimidine-loaded liposomes with the plasmin-
binding tripeptide. The physicochemical properties (size,
structure, surface) of pyrazolo[3,4-d]pyrimidine-loaded bare
liposomes (LP) and pyrazolo[3,4-d]pyrimidine-loaded tripep-
tide-conjugated liposomes (TLP) will be examined and
compared. Finally, the in vitro uptake profiles and cytotoxicity
of free drugs and the drugs entrapped in liposomes with and
without plasmin-binding tripeptide were examined on the
HepG2 HCC cell line.
Synthesis and characterization of pyrazolo[3,4-d]pyrimidine
compounds (S29, SI113, SI163) were previously reported by
us.10,35,36 Structures of compounds have been reported in Figure
1, as well as the tripeptide-linker (TRP) structure. ADME
properties, activity against Src and Sgk1, and IC50 against HepG2
cells of S29, SI113, and SI163 have been reported in Table 1.
The tripeptide-linker (TRP) was designed and synthesized in
good yields, developing a simple liquid phase method-based
procedure. In order to synthesize the tripeptidyl portion, it was
pivotal to set up an orthogonal protection strategy. The synthetic
pathway is summarized in Scheme 1. H-Lys(Z)-OH methylester
hydrochloride, obtained by treatment of H-Lys(Z)-OH (1) with
SOCl2 in MeOH, was coupled with Boc-Phe-OH using EDC·
HCl/DMAP as an activating system. Dipeptide 3 was then N-
deprotected with 10% TFA affording 4, which was coupled with
Boc-D-Ala-OH in the presence of EDC·HCl/DMAP. The
carbobenzyloxyl moiety was then cleaved by hydrogenation. In
B
DOI: 10.1021/acsmedchemlett.8b00062
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Letter
a
Scheme 1. Preparation of Tripeptide Portion D-Ala-Phe-Lys
Figure 2. Schematic representation of TRP (R1 = CH3; R2 = CH3−Bn;
R3 = CH3−(CH2)4−NH2) and the sites that undergo MS/MS
fragmentation, their m/z (black), and the corresponding collision
energy in volts (red).
a
Reagents and conditions: (a) SOCl2, MeOH, r.t., 16 h; (b) Boc-Phe-
OH, DMAP, EDC·HCl, CH2Cl2 dry, r.t., 16 h; (c) 10% TFA, CH2Cl2
dry, r.t., 1 h; (d) Boc-D-Ala-OH, DMAP, EDC·HCl, CH2Cl2 dry, r.t.,
16 h; (e) H2, Pd/C 10%, MeOH, HCl conc., r.t., 12 h; (f) Boc2O,
THF, 5% NaOH, r.t., 16 h.
be fragmented and produced ions with m/z 276.17, 353.20,
219.11, 206.13, and 129.10. The 191.12 and 100.07 m/z
fragments derived from the rupture of the same bond, as well
as 219.11 and 206.13. Details about the structures of the
fragments are reported in the Supporting Information.
conclusion, after pH adjustment to 5, the reaction of the
tripeptidyl intermediate 6 with Boc2O and 5% NaOH led to the
formation of the desired tripeptide D-Ala-Phe-Lys (7).
Therefore, a linker H2N−CH2−CH2−SH was introduced at
the C-terminal tripeptide sequence by amide bond formation in
order to activate the peptide toward maleimide liposomes
(Scheme 2). The 2-aminoethanethiol (8) hydrochloride
S29, SI113, and SI163 were chosen from the wide pyrazolo-
[3,4-d]pyrimidine derivatives library to be formulated as
targeting liposomes against HCC. Ki values for Src in the
submicromolar range and ADME parameters have been the main
factors determining the selection. Particular focus has been paid
to SI113 because of its inhibitory activity against both c-Src and
Sgk1, its effectiveness in inducing apoptosis in HepG2 cell line
and in reducing tumor growth in HepG2 xenografted mice.14
With the aim of improving their ADME profile and enhancing
their therapeutic effectiveness, a stealth liposomal formulation
has been developed. S29-LP, SI113-LP, and SI163-LP have been
synthesized by thin layer evaporation method from DPPC/
CHOL/DPPE-PEG2000, 2:1:0.1 molar ratio. Each compound
(S29, SI113, SI163) was added to the lipid mixture to obtain a
final concentration of 0.8 mM in the final volume. The dry lipid
film was hydrated in 25 mM HEPES and 140 mM NaCl buffer,
pH 7.4, at 60 °C. The downsizing of large multilamellar vesicles
(MLV) was achieved using sonic energy, reducing dimension
and lamellarity to produce small unilamellar vesicles (SUV). The
liposomal suspensions were filtered through 0.2 μm filters to
exclude oversized liposomes that may undergo more rapid and
extensive opsonization after intravenously injection, inducing
their elimination from the blood circulation by the mononuclear
phagocyte system (MPS).37 Mean diameters, ζ-potentials, and
PDI were measured by dynamic light scattering (DLS), and
results are shown in Table 2. Size and ζ-potential values for S29-
LP, SI113-LP, and SI163-LP were 153.7, 111.6, 119.3 nm and
−29.7, −47.7, −38.7 mV, respectively. These results suggest that
the obtained liposomes were stable and did not have the
tendency to form aggregates, increasing in dimensions, thus
being optimal for in vivo administration. Indeed, ζ-potential
values lower than −25 mV or greater than +25 mV indicate an
electrostatic repulsion sufficient to maintain separation between
particles.38 Drug encapsulation efficacy % was determined by
LC−UV−MS (for methods, see Supporting Information) after
the lysis of liposomes by adding ethanol to the suspensions, and
results have been reported in Table 2. EE% ranged from 44%
(S29) to 83% (SI163) and 95% (SI113). S29 showed a lower EE
% probably as a consequence of its high hydrophobicity and of
the competition with cholesterol for the hydrophobic space in
the lipid bilayer.39
a
Scheme 2. Preparation of TRP
a
Reagents and conditions: (g) 3,4-dihydro-2H-pyran, p-TsOH cat,
CH2Cl2 dry r.t., 5 h; (h) D-Ala-Phe-Lys (7), DMAP, EDC·HCl,
CH2Cl2 dry, r.t., 16 h; (i) 10% TFA, CH2Cl2 dry, r.t., 16 h.
commercially available was SH-protected with 3,4-dihydro-2H-
pyran (9). After, linker 9 was coupled with tripeptide 7 using the
same condensing agents reported above. Finally, all protecting
groups were removed by a single step with 10% TFA providing
the final product TRP (11). This approach allowed preparing
tripeptide-conjugated liposomes by coupling the free thiol group
of TRP and the maleimide terminus of DSPE-PEG2000-
maleimide.
Further details regarding materials, synthesis, and NMR
characterization of TRP are reported in the Supporting
Information.
TRP was ionized and fragmented by LC−MS/MS with
progressively higher collision energy to produce spectra that
were used for its identification and characterization. In Figure 2, a
schematic representation of the TRP sites that undergo MS/MS
fragmentation is illustrated. Being the weakest, peptide bonds
CO−NH require low collision energy (−20, −15, and −10 V) to
C
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Table 2. Properties of Bare Liposomes and TRP-Conjugated Liposomes Encapsulating Pyrazolo[3,4-d]pyrimidines
formulation
S29-LP
S29-TLP
SI113-LP
SI113-TLP
SI163-LP
SI163-TLP
a
EE %
44 7.5
153.7 5.4
0.679 0.034
−29.7 3.48
1.6 1.2
31 8.3
103.4 6.67
0.183 0.012
−20.1 4.36
95 1.5
111.6 12.6
0.539 0.058
−47.7 4.79
8.0 3.5
94 3.2
118.3 6.7
0.228 0.043
−36.3 5.25
2.5 1.9
83 6.5
119.3 6.5
0.813 0.114
−38.7 4.28
1.8 1.4
98 1.2
121.9 11.76
0.317 0.084
−15.4 6.12
1.0 1.1
b
size (nm)
b
PDI
b
ζ potential (mV)
IC50 (μM)
1.8 1.1
a
b
For experimental details see Supporting Information. Determined by DLS.
Figure 3. Left: Fluorescence microscopy images of HepG2 cells following LPs and TLPs administration: cells after 5 min of incubation with LPs (A) and
TLPs (B) and cells after 1 h of incubation with LPs (C) and TLPs (D). Green, liposomes; red, cell marker; blue, nuclei. Right: Relative fluorescence
intensity per cell area histograms for each formulation.
S29-TLP, SI113-TLP, and SI163-TLP dried lipid films have
been prepared by the method previously described for LPs,
except for the addition of DSPE-PEG2000-maleimide to the lipid
mixture (DPPC/CHOL/DPPE-PEG2000/DSPE-PEG2000-
Mal, 2:1:0.08:0.02 molar ratio). After the preparation of the
lipid film and its hydration, the sonication and the filtration of the
liposomal suspensions, surface decoration of liposomes was
achieved by coupling the linking portion of the tripeptide to the
maleimide terminus of PEG chains laying on the outer liposomal
membrane. The coupling reaction between liposomes and TRP
was run at a molar ratio of TRP to DSPE-PEG2000-Mal of 1:4
overnight at 4 °C. The amount of TRP conjugated to the
liposomal membrane was determined after the lysis of liposomes
by subtracting free TRP from the initial TRP concentration. The
33% of the total amount of TRP effectively bound the liposomal
surface, and about 300 tripeptide molecules per liposomes were
estimated. Uncoupled TRP was separated from the liposomal
suspension by dialysis. TRP-liposomes have been characterized
by DLS and HPLC-MS without remarkable differences with
respect to bare liposomes (Table 2). Membrane ζ-potential
values were measured to be −20.1 mV for S29-TLP, −36.3 mV
for SI113-TLP, and −15.4 mV for SI163-TLP. Membrane ζ-
potentials for each TLP increased with respect to the
corresponding LP. This enhancement is due to the positive net
charge possessed by the tripeptide that induces a lower
electrostatic repulsion between particles. Nevertheless, particle
sizes were measured to be 103.4 nm for S29-TLP, 118.3 nm for
SI113-TLP, and 121.9 nm for SI168-TLP, which are excellent
dimensional parameters for a future intravenous administration.
Moreover, PDI values demonstrated an improvement in
homogeneity and uniformity of particles size distribution of
TRP-liposomes with respect to TRP free-liposomes. Indeed,
TRP-liposomes showed a PDI around 0.2−0.3, indicating a
relatively monodispersed particle population, whereas TRP free-
liposomes showed PDI values >0.5, suggesting the presence of a
broad particle size distribution. Encapsulation efficacy % was
greater than 90% for SI113 and SI163 TRP-liposomal
formulations (94% and 98%, respectively) and 31% for S29-TLP.
The in vitro antiproliferative effects of anticancer pyrazolo[3,4-
d]pyrimidines-loaded TRP-conjugated liposomes, the respective
bare liposomes, and free drugs dissolved in DMSO were
evaluated by cytotoxicity assay and determination of cell survival
rates by using HepG2 HCC cell line (for materials and methods,
see Supporting Information). Results, shown in Tables 1 and 2,
indicate that S29 activity is not significantly altered when the
compound is delivered by liposomes, both TRP-conjugated and
TRP-free, while SI113 and SI168 displayed a higher cytotoxic
activity when delivered by liposomes compared to free
compounds. Moreover, for these two molecules, the presence
of the tripeptide on the liposomal membrane surface increased
the activity of the liposomes-delivered compounds: SI113-TLP
resulted 3 times more active than SI113-LP and 5 times more
than free compounds, and the cytotoxicity of SI163-TLP was
almost 2 times greater than SI163-LP and 3 times greater than
free SI163. All the free compounds and the respective liposomal
formulation did not show cytotoxicity in normal cells (RWPE-1
cell line) in the concentration range of their IC50 values for
HepG2. The only formulation that provoked a cellular mortality
greater than 20% was TRP-liposomal S29. Cytotoxicity of empty
TRP-liposomes and bare liposomes was evaluated on HepG2,
and both were proved to be safe. These outcomes proved that the
intracellular delivery of the anticancer pyrazolo[3,4-d]-
pyrimidines was enhanced by plasmin-binding tripeptide-
conjugated liposomes with an increased amount of drug available
to act against cancer cells. The fact that S29 activity was not
enhanced by the inclusion of the molecule in the liposomal
structure is probably due to the poor encapsulation efficacy %
that results in a lower concentration of compound per single
carrier.40 Both for the low EE% and the cytotoxicity tests results,
S29 has been considered unpromising, and it will not be selected
for future studies.
In vitro cellular uptake assay was performed. Fluorescent TRP-
liposomes and bare liposomes were prepared, incorporating a
fluorescent phospholipid (PE-CF) in the lipids mixture and
subsequently administered to HepG2 cells (see Supporting
Information).
D
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ORCID
After 5 min and 1 h from the administration, cells treated by
both the liposomal formulations were observed by confocal
fluorescence imaging. Z-stack images and histograms indicating
relative fluorescence intensity per cell area for each formulation
are shown in Figure 3 (fluorescence values have been
extrapolated from the images by the software ImageJ): HepG2
cells treated with TRP-liposomes displayed a higher fluorescence
(Figure 3B,D) than the cells treated with free TRP liposomes
(Figure 3A,C). Images suggest that the presence of the PM-
binding tripeptide on the liposomal surface allowed an improved
and more rapid interaction with the cellular membrane and
subsequent internalization, confirming the evidence that a
greater amount of anticancer drug was taken up by the cells,
being more effective.
Elena Dreassi: 0000-0001-8987-940X
Mario Chiariello: 0000-0001-8434-5177
Maurizio Botta: 0000-0003-0456-6995
Author Contributions
○These authors contributed equally. P.C. and A.A., cellular and
confocal microscopy studies; G.I. and S.S., synthesis and
characterization of compounds; C.Z., E.D., and A.M., MS/MS
spectrometry studies; C.Z., F.C., and A.M., liposomes prepara-
tion and characterization; P.C., G.I., C.Z., E.D., M.C., S.S., M.B.,
and A.M., design of methods and experiments, analysis and
interpretation of data; G.I., M.B., and A.M., preparation of the
manuscript. All authors have given approval to the final version of
the manuscript.
In summary, first we synthesized and characterized a tripeptide
with a specific sequence able to bind cell-associated PM that is
proven to be overexpressed in HCC cells. After full character-
ization of TRP via collision-induced dissociation by tandem mass
spectroscopy, we developed three liposomal carrier systems that
were encapsulated with a pyrazolo[3,4-d]pyrimidine compound
each (S29, SI113, SI163) and decorated with the PM-binding
tripeptide, with a density of ∼300 molecules of tripeptide per
liposome. Encapsulation efficacy % was greater than 90% for
SI113 and SI163 formulations and 31% for S29; particles size was
measured to be around 100 nm for all the formulations and
membrane ζ-potential values ranged from −36.3 to −15.4 mV. In
general, all the tripeptide-liposomal formulations have shown no
remarkable differences in characterization profiles with respect to
the corresponding bare liposomes (except for an enhancement in
ζ-potential values due to the positive charge of TRP and an
improvement in PDI values) and present good parameters for
future in vivo administration. The in vitro antiproliferative effects
of anticancer pyrazolo[3,4-d]pyrimidines-loaded tripeptide-
conjugated liposomes were then evaluated on HepG2 cells and
both SI113 and SI163 tripeptide-liposomes resulted more active
than bare liposomes and free compounds. This outcome suggests
an enhancement in cell-penetrating ability provided by the
presence of the tripeptide on the liposomal surface and
consequently a greater amount of drug delivered into the cancer
cells, which suppressed proliferation and survival. This evidence
was confirmed by confocal microscopy tests that showed a
greater and more rapid cellular internalization, promoted by the
interaction between the tripeptide and cell surface-associated
PM. All these results taken together represent a very promising
starting point for future in vivo pharmacokinetic and
pharmacodynamic studies.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was supported by the Italian Association for Cancer
Research AIRC IG2015-17677 grant and Lead Discovery, Siena
s.r.l.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00062.
Reagents, synthesis, NMR, LC−MS and MS/MS
characterization of TRP; materials and experimental
details for preparation and characterization of liposomes
and for cellular assays, and instruments details (PDF)
(10) Rossi, A.; Schenone, S.; Angelucci, A.; Cozzi, M.; Caracciolo, V.;
Pentimalli, F.; Puca, A.; Pucci, B.; La Montagna, R.; Bologna, M.; Botta,
M.; Giordano, A. New pyrazolo-[3,4-d]-pyrimidine derivative Src kinase
inhibitors lead to cell cycle arrest and tumor growth reduction of human
medulloblastoma cells. FASEB J. 2010, 24, 2881−92.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +39-0577-234306. Fax: +39-0577-234303. E-mail: botta.
maurizio@gmail.com.
E
DOI: 10.1021/acsmedchemlett.8b00062
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