Folic acid-conjugated europium complexes as luminescent probes for selective targeting of cancer cells

Article abstract of DOI:10.1021/jm501945w

We report the synthesis of three optical probes (Eu³⁺1, Eu³⁺2, and Eu³⁺3) having a luminescent Eu complex (signaling unit) bonded in different positions to folic acid (FA), the folate receptor (FR) targeting unit. The structures of the two regioisomers Eu³⁺1 and Eu³⁺2 were assigned by mass spectrometric experiments. The optical properties and stability of these probes were assessed in phosphate-buffered saline, cell culture medium, rat serum, and cellular lysate, and results indicated that they are chemically and photophysically stable. Cytotoxicity was studied with ovarian cancer cells having high (SKOV-3), intermediate (OVCAR-3), low (IGROV-1), or null (A2780) expression of FRs. The internalized probe, evaluated in SKOV-3, IGROV-1, and A2780 cells, was in the order Eu³⁺2 > Eu³⁺1 > Eu³⁺3. No internalization was observed for A2780 cells. Such results, together with those obtained in competition experiments of FA versus Eu³⁺2 and FA or Eu³⁺2 versus ³H-FA, indicate that internalization is receptor-mediated and that Eu³⁺2 shows high selectivity and specificity for FR.

Full text of DOI:10.1021/jm501945w

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Article
Folic Acid-Conjugated Europium Complexes as
Luminescent Probes for Selective Targeting of Cancer Cells.
Silvio Quici, Alessandro Casoni, Francesca Foschi, Lidia Armelao, Gregorio Bottaro, Roberta
Seraglia, Cristina Bolzati, Nicola Salvarese, Debora Carpanese, and Antonio Rosato
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501945w • Publication Date (Web): 20 Jan 2015
Downloaded from http://pubs.acs.org on January 28, 2015
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Folic AcidꢀConjugated Europium Complexes as
Luminescent Probes for Selective Targeting of
Cancer Cells.
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†
†
†
‡
‡
Silvio Quici,* Alessandro Casoni, Francesca Foschi, Lidia Armelao, Gregorio Bottaro,
#
# �
§
§
Roberta Seraglia, Cristina Bolzati, Nicola Salvarese, Debora Carpanese and Antonio
§
☼
Rosato.
†
Istituto di Scienze e Tecnologie Molecolari (ISTM), Consiglio Nazionale delle Ricerche
‡
(
CNR), Via C. Golgi 19, 20133 Milano, Italy, Istituto per l'Energetica e le Interfasi (IENI),
Consiglio Nazionale delle Ricerche (CNR) e Consorzio Interuniversitario per la Scienza e
Tecnologia dei Materiali (INSTM), Dipartimento di Scienze Chimiche, Università di Padova,
#
Via Marzolo 1, 35131 Padova, Italy, Istituto per l'Energetica e le Interfasi (IENI), Consiglio
�
Nazionale delle Ricerche (CNR), Corso Stati Uniti 4, 35127 Padova, Italy, Dipartimento di
§
Scienze Farmaceutiche, Università di Padova, Via Marzolo 5, 35131 Padova, Dipartimento di
Scienze Chirurgiche Oncologiche e Gastroenterologiche, Università di Padova, Via Giustiniani
☼
2
, 35128 Padova, Italy and Istituto Oncologico Veneto IOVꢀIRCCS, Via Gattamelata 64,
35128 Padova, Italy.
KEYWORDS. folic acidꢀconjugates; luminescent antenna complexes; ovarian cancer; molecular
targeting; optical imaging.
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ABSTRACT. We report the synthesis of three optical probes (Eu ⊂1, Eu ⊂2 and Eu ⊂3)
having a luminescent Euꢀcomplex (signalling unit) bonded in different positions to folic acid
3+
(
FA), the folate receptor (FR) targeting unit. The structure of the two regioisomers Eu ⊂1 and
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Eu ⊂2 was assigned by MS experiments. The optical property and stability of these probes were
assessed in PBS, cell culture medium, rat serum and cellular lysate and results indicated that they
are chemically and photophysically stable. Cytotoxicity was studied with ovarian cancer cells
having high (SKOVꢀ3), intermediate (OVCARꢀ3), low (IGROVꢀ1) or null (A2780) expression
of FRs. The internalized probe, evaluated in SKOVꢀ3, IGROVꢀ1 and A2780 cells, was in the
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+
3+
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order Eu ⊂2 > Eu ⊂1 > Eu ⊂3. No internalization was observed for A2780 cells. Such
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results, together with those obtained in competition experiments of FA vs. Eu ⊂2 and FA or
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+
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Eu ⊂2 vs. HꢀFA, indicate that internalization is receptorꢀmediated and that Eu ⊂2 shows a
high selectivity and specificity for FR.
INTRODUCTION. In the modern medicine, and particularly in cancer, a pivotal role is played
by theranostic systems containing intimately coupled both a targeted therapeutic drug and an
imaging moiety, which selectively accumulate within diseased cells and tissues and allow their
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monitoring before, during and after therapy. The development of theranostics would not have
2
been possible without the great advances made in the field of molecular imaging, and in the
3
synthesis of efficient targeting moieties.
Over the years, several imaging techniques have been refined, including magnetic resonance
imaging (MRI), single photon emission computed tomography (SPECT), positron emission
4
tomography (PET) and optical imaging (OI). Although the heterogeneity of tissues limits the
transparency window of the human body and generally attenuates the signal intensity due to
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absorption and scattering by endogenous chromophores, fluorescence intensity imaging (FII) is
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ideally as sensitive as nuclear imaging, cost effective and widely available. Luminescence
measurements show submicron spatial resolution and very fast (from nano to millisecond)
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temporal resolution, making it possible to detect biological events at an early stage and to
change more rapidly the therapeutic treatment of the disease based on data obtained. For
example, fluorescent imaging allowed intraoperative detection of tumor in real time, providing a
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direct guidance for surgeons for an effective and complete resection of the tumor. Moreover,
optical methods show their effectiveness when a simultaneous multispectral analysis of different
probes during the same operation is required, a practice that would be impossible with other
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inherently monochromatic imaging methods.
The use of luminescent complexes in cell imaging is particularly appealing mostly because of
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the peculiar properties of the luminescence emission from the metal excited states. In this
context, a leading role is played by the luminescent lanthanide complexes (LLCs) that show a
series of potential advantages: 1) narrow and intense bands which ensure high color purity, large
Stokes shifts with no overlapping of excitation and emission spectra, long emission lifetimes
enabling time resolved measurements; 2) the loading of LLCs can be increased since they do not
show luminescence selfꢀquenching that is a very strong drawback for organic dyes; 3) different
LLCs can be prepared from the same ligand so that, depending on the antenna, they can be
excited by the same wavelength; 4) LLCs show very high chemical, thermal, kinetic and
3+
photophysical stability; 5) very low toxicity compared with the corresponding Gd complexes of
diethylentriaminetetraacetic acid (DTTA) and of 1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriacetic
1
0
acid (DO3A) widely used in vivo as contrast agents for MRI.
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A second aspect that must be addressed in the development of tumorꢀspecific imaging agents is
represented by the choice of the most appropriate carrier. Compared to other molecular vectors
such as proteins, DNA, monoclonal antibodies and peptides, commonly used for selective drug
and/or imaging agent delivery in tumor cells and diseased tissues, folic acid (FA) shows some
peculiar features, i.e. low cost, high stability in a range of temperature and pHs, compatibility
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with aqueous and organic solvents and easy conjugation synthetic protocols. In addition, this
nonꢀimmunogenic target ligand possesses high binding affinity (K = 100 pM) with folate
d
receptors (FRs), whose expression in physiological conditions is largely restricted to cells
important for the embryonic development and folate resorption (kidney). FRs are a family of
glycoproteins (35ꢀ40 kDa) and are divided in three subtypes: FRα and FRβ, which are linked to
the cell membrane via glycosylꢀphosphatidilinositol (GPI) anchors, and FRγ, that is freely
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soluble since it lacks the GPI fragment.
Among these, the FRα isoform is the most widely expressed on cell membrane of many
epitelial tumors, with very low expression in normal tissues, whereas FRβ is upregulated on
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activated macrophages that are responsible of inflammatory and autoimmune diseases. The
mechanism of transport of FRs, although not completely elucidated, seems to involve the
activation of protein by the FA (or folate conjugated) interaction and subsequent internalization
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through receptorꢀmediated endocytosis.
FA consists of three distinct portions, namely a pterin moiety, a pꢀaminobenzoate linker and a
glutamate residue. This molecular structure contains at least five possible sites for
functionalization, which are potentially useful for the preparation of folate conjugates (Figure 1).
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Figure 1. Chemical structure of folic acid (arrows indicate the possible sites of conjugation) and
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of Eu ⊂1, Eu ⊂2 and Eu ⊂3
Nowadays, the choice of the conjugation site able to retain optimal receptor binding properties
is still under discussion. Almost all the papers appeared in the literature report the
functionalization of the carboxyl groups of the FA glutamic acid residue by reaction with an
amino derivative. This reaction affords a mixture of two regioisomers (αꢀ and γꢀsubstituted) that,
in some cases, is used as such in the cellular uptake experiments, since the separation of the pure
isomers can be quite difficult to achieve. Although many authors state that FA must be
substituted in the γ position and that it is essential to leave the α carboxyl group free to maintain
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high recognition selectivity, other authors claim that the functionalization position has no effect
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on the binding ability of FA conjugates and disprove the necessity of a free carboxyl group on
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the glutamate moiety for the FR binding. Only recently the study of the crystal structure of FA
bound to human FR has evidenced a preferential interaction of pterin head group with the
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receptor’s binding pocket.
During the years, a wide number of FAꢀbased conjugates have been synthesized and tested
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either in vitro or in vivo. These include protein toxins, low molecular weight drugs,
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radiolabelled drugs for imaging and therapy, liposome entrapped drugs, and nanoparticles.
It must be pointed out that, once linked to FA, almost any type of cargo can be specifically
driven inside diseased cells overexpressing FRs. Among these, low molecular weight FA
conjugates (< 2000 Da), in general, offer excellent dynamic characteristics in terms of tissue
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penetration and clearance.
To our knowledge, there is only one paper in the literature in which a luminescent folateꢀ
bioconjugate formed by a trifluoromethylphenylacetylacetonate europium complex attached to
FAꢀappended phenanthroline has been prepared and used as targeted luminescent probe for HeLa
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cancer cells. Authors reported that the observed cellular uptake (about 4x10 bioconjugate
molecules/cell) was due to FR binding since the uptake strongly decreased in the presence of
exogenous folic acid. However, the characterization of the system was poor and no data about
the cytotoxicity and stability of the system was reported. A series of highly luminescent Eu(III)
complexes for the optical imaging of subcellular compartments, such as lysosomes, mitochondria
25
and endoplasmic reticulum of various mammalian cells was recently reported by Parker et al.
To study the relationship between the conjugation site and the recognition capability of the
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+
resulting conjugates by FR, here we report the synthesis of three new optical probes Eu ⊂1,
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+
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Eu ⊂2 and Eu ⊂3 (Figure 1) that are characterized by a luminescent europium antenna
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complex (signalling unit) covalently linked to FA (targeting unit). In these probes, the signalling
unit is connected to different positions of the FA structure, namely the α and γ carboxylic groups
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of the glutamic residue for Eu ⊂1 and Eu ⊂2 respectively, and the nitrogen atom of the pꢀ
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aminobenzoic residue for Eu ⊂3. The two regioisomers Eu ⊂1 and Eu ⊂2, obtained as a
mixture from the preparation procedure, were carefully separated as pure isomers and identified
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+
by mass spectrometry experiments. Eu ⊂3 was obtained as a unique compound through a
multistep synthesis. All three optical probes were thoroughly investigated for photophysical
properties in different biological media, and evaluated for cytotoxicity and cellular uptake on
ovarian cancer cell lines having different FR expression.
RESULTS AND DISCUSSION
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Synthesis of bioconjugate complexes. Conjugates Eu ⊂1 and Eu ⊂2 were obtained as a
mixture of α and γ regioisomers by condensation of commercially available folic acid with 10ꢀ
[
4ꢀ(3ꢀaminopropyloxy)benzoylmethyl]ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriacetic
acid
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+
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europium complex (Eu ⊂4) carried out in a 4:1 (v/v) mixture of anhydrous DMSO and
pyridine as solvent and dicyclohexylcarbodiimide (DCC) as condensing agent (Scheme 1).
A careful analysis by mass spectrometry of the two pure regioisomers isolated by HPLC
3+
3+
allowed us to assign their structures. The ESI mass spectra of compounds Eu ⊂1 and Eu ⊂2
(see SI) are characterized by the presence of the cluster ions at m/z 1111, corresponding to
+
[
M+H] . The presence of europium within the molecule is proved by the typical isotopic cluster.
To distinguish the two possible structures of the compounds, MS/MS experiments were
+
performed on the monoisotopic [M+H] ions at m/z 1109. The MS/MS spectra related to the ion
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at m/z 1109 of the two compounds were different, leaving aside the common losses of H O and
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CO which give rise to the ions at m/z 1091 and 1065, respectively (Figure 2).
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Scheme 1. Synthesis of Eu ⊂1 and Eu ⊂2
+
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Figure 2. MS/MS spectra of monoisotopic [M+H] ions at m/z 1109 of Eu ⊂1 (a) and Eu ⊂2
(b)
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In the case of Eu ⊂1, the most favored decomposition pathway leads to the formation of ion
3+
at m/z 934, corresponding to the loss of 2ꢀaminoꢀ6ꢀmethylꢀ1Hꢀpteridinꢀ4ꢀone. For Eu ⊂2, the
most favored decomposition pattern leads to the formation of the ion at m/z 686 corresponding to
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the protonated Eu ⊂4. Moreover, in the MS/MS spectrum of ion at m/z 1109 of Eu ⊂1 other
peaks are present such as those at m/z 797 and 752 that are completely absent in the case of
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compound Eu ⊂2. These peaks can be reasonably explained by rearrangements originating from
the decomposition of the ion at m/z 916, corresponding to the loss of water from the ion at m/z
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34 (for detailed fragmentation pathway see SI). It is to note that the cyclization completely
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inhibits the rearrangement of the amidic bond observed in the case of compound Eu ⊂2.
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The synthesis of Eu ⊂3 derivative was carried out by a multiꢀstep procedure reported in
Scheme 2, which required as last step a Huisgen cycloaddition between the protected Nꢀ
propargyl folate 11 and 10ꢀ[4ꢀ(3ꢀazidopropoxy)benzoylmethyl]ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ
3+
1
,4,7ꢀtriacetic acid europium complex (Eu ⊂18) under standard conditions as reported by
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7
Mastarone et al. , followed by the deprotection under basic conditions and at room temperature
3+
of both amine and carboxyl groups, to afford Eu ⊂3 in 81% yield after purification by column
chromatography with Amberlite XAD 1600T.
The synthesis of the protected Nꢀpropargyl folate 11 was conducted according to Scheme 2. At
first, Nꢀ(6ꢀ(bromomethyl)ꢀ4ꢀoxoꢀ1,4ꢀdihydropteridinꢀ2ꢀyl)acetamide (8) was prepared in a threeꢀ
step procedure by condensation of commercially available 6ꢀhydroxyꢀ2,4,5ꢀtriaminopyrimidine
sulfate (5) with methylglyoxal in reducing media by Na SO /Na S O at 0°C to give 2ꢀaminoꢀ6ꢀ
2
3
2
2
5
methylpterin 6, followed by acylation with acetic anhydride in acetic acid at 140°C, and finally
by bromination of the acetamido derivative 7 with Nꢀbromosuccinimide (NBS) in acetic acid at
reflux in the presence of dibenzoylperoxide affording the desired intermediate 8 in satisfactory
yield (Scheme 2).
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Scheme 2. Synthesis of Eu ⊂3
Commercially available Nꢀ(4ꢀaminobenzoyl)ꢀLꢀglutamic acid diethyl ester (9) was reacted
with propargyl bromide in anhydrous DMA in the presence of K CO solid as base to afford the
2
3
propargylated derivative 10. The condensation of compound 10 with the bromomethylꢀpterinyl
acetamide 8 was carried out in DMA at 95 °C and CaCO solid as base affording the fully
3
protected Nꢀpropargyl folate 11 in good yield after purification by column chromatography.
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The synthesis of europium complex Eu ⊂18 was carried out as outlined in Scheme 3.
Reaction of commercially available 4ꢀhydroxyacetophenone (12) with 1,3ꢀdibromopropane was
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carried out in CH CN at reflux for 24 h, in the presence of solid Na CO as base affording 4ꢀ(3ꢀ
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bromopropoxy)ꢀacetophenone (13).
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44
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52
53
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60
3
+
Scheme 3. Synthesis of Eu ⊂18
This product was reacted with sodium azide in anhydrous DMSO at room temperature for 24 h
2
8
to give the azido substituted derivative 14 in excellent yield. Treatment of 14 with NBS and pꢀ
toluenesulfonic acid monohydrate in CH CN at reflux for 2 h produced 4ꢀ(3ꢀ
3
azidopropoxy)phenacylbromide 15 with a 80% yield after purification by column
2
9
chromatography. Alkylation of 1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriacetic acid tertꢀbutyl
2
7, 30
ester hydrobromide 16
with 15 was carried out in CH CN at reflux in the presence of
3
Na CO as base, and afforded sodium bromide complex 17 isolated in quantitative yield after
2
3
column chromatography. Indeed, the ability of alkylated DO3A triesters to coordinate sodium
31
cation has been already assessed and the presence of the sodium bromide complex simplifies
the chromatographic purification and allows higher yields of isolated product.
Decomplexation takes place during deprotection of the tertꢀbutyl groups that was achieved by
basic hydrolysis of 17 carried out with NaOH in aqueous methanol at room temperature to afford
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the corresponding triacid derivatives 18 in quantitative yield. It must be pointed out that the
azido group of 17 is unstable with CF COOH in anhydrous CH Cl at room temperature, which
3
2
2
are the classical conditions used for the deprotection of the tertꢀbutyl ester groups. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
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8
9
0
1
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3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
3+
preparation of the corresponding europium complex Eu ⊂18 was carried out by mixing
stoichiometric amounts of EuCl · 6H O and ligand 18 at reflux for 16 h and adjusting the pH at
3
2
6
.5ꢀ7.0 with 2% aqueous NaOH. The product was purified by an Amberlite XAD 1600T column
to afford 89% of a pale beige crystalline solid (detailed experimental procedures are reported in
SI).
3
+
3+
3+
Photophysical characterization. The absorption spectra of Eu ⊂1, Eu ⊂2, Eu ⊂3, FA and
1
0ꢀ[4ꢀ(3ꢀaminopropyloxy)benzoylmethyl]ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriacetic acid
3
+
europium complex (Eu ⊂4) are displayed in Figure 3.
3+
3+
3+
ꢀ5
Figure 3. Absorption spectra of Eu ⊂1, Eu ⊂2, Eu ⊂3 in 10 M water solutions. The
3
+
absorption spectra of FA and of the europium complex Eu ⊂4 in H O are displayed as dotted
2
curves
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The absorption spectrum of FA is characterized by the presence of two bands in the 250ꢀ500
nm range. These absorptions can be attributed to electronic transitions localized on the pterin
moiety of FA. The band centered at 285 nm overlaps with those of pꢀaminobenzoate residue of
10
11
12
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14
15
16
17
18
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32
FA. All the absorption features of the folic acid are clearly visible in the absorption spectra of
3
+
3+
3+
Eu ⊂1, Eu ⊂2 and Eu ⊂3. Furthermore, in the europium folate complexes the absorption
band located between 250 and 350 nm is broadened due to a partial overlap between folic acid
and acetophenone absorption bands, the latter being centered at 310 nm in the starting europium
3+
33
complex Eu ⊂4.
ꢀ5
3+
3+
3+
Under 310 nm excitation, 10 M water solutions of Eu ⊂1, Eu ⊂2 and Eu ⊂3 appear bright
red due to the sensitized emission of europium. The emission spectra of europium folate
complexes are composed of several narrow bands in the 570 ꢀ 710 nm range associated with the
3
+ 5
7
Eu D → F transitions (J = 0 ꢀ 4): the higher the J value, the lower the transition energy
0
J
(
Figure 4a).
3
+
3+
3+
ꢀ5
Figure 4. Emission spectra of Eu ⊂1, Eu ⊂2 and Eu ⊂3 in water solutions 10 M excited at
3
+
3
10 nm (a); excitation spectra of Eu ⊂1 monitored at 440 and 615 nm, and of FA monitored at
440 nm (b)
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5
5
6
Europium emission shows the same profile as that observed for the starting europium complex
3
+
Eu ⊂4. In the blueꢀgreen region (λ < 550 nm), all europium folate complexes have a different
3+
behavior. In particular, in the emission spectra of Eu ⊂1 a broad emission centered at 440 nm is
0
1
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8
9
0
1
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8
9
0
1
2
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8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
clearly visible. This emission is also present in the other derivatives although with lower
3
+
intensity, and reaches the minimum for Eu ⊂3. The nature of this emission has been identified
through photoluminescence excitation (PLE) experiments (Figure 4b). The PLE spectra of
3
+
Eu ⊂1 and folic acid monitored at 440 nm have a similar shape indicating that the emission
band is originated from electronic transitions localized on the folic acid residue. It is interesting
to note that the excitation spectrum for europium (λem = 615 nm) consists of a single band with
maximum at 310 nm, which overlaps the absorption spectrum of acetophenone. These findings
exclude the participation of folic acid in the sensitization of europium emission. It is worth to
3
+
3+
highlight that Eu ⊂2 and Eu ⊂3 have the same behavior.
The photophysical data of the various complexes are summarized in Table 1. The pristine
3
+
Eu ⊂4 has a photoluminescence quantum yields (PLQY) of 7% despite of the presence of a
water molecule in the first coordination sphere. As a consequence of the conjugation with folic
3
+
3+
3+
acid a lower value has been detected for the PLQY of Eu ⊂1, Eu ⊂2 and Eu ⊂3. It is likely
that the partial overlap between the absorption spectra of acetophenone and folic acid could
affect the PLQY since during excitation less photons are available for lanthanide sensitization.
Lifetimes and number of coordinated water molecule are not affected by the presence of FA.
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3+
ꢀ5
Table 1: Photophysical data for the complexes Eu ⊂X (X = 1, 2, 3 and 4), 10 M in water.
a,c
a,c
d
ꢀ
1
ꢀ1 a
a,b
τ
H2O (ms)
τD2O (ms)
q
Complex
ε (M cm )
PLQY
0.07
0.01
0.02
0.01
3
+
Eu ⊂4
19100
0.61
0.59
0.59
0.61
2.2
1.9
1.9
2.1
1
10
11
12
13
14
15
16
17
18
19
20
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33
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54
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59
60
3
+
Eu ⊂1
30500
1
1
1
3
+
Eu ⊂2
32100
3
+
Eu ⊂3
38900
a
b
Measured at 310 nm. Three independent measurements were carried out on each complex
with an estimated error of ± 20%. Estimated error ± 10%. Number of water molecules in the
first coordination shell estimated by the Horrocks formula: q = 1.11(1/τ
c
d
3
4
ꢀ 1/τ
ꢀ 0.31).
D O
2
Eu
H O
2
The luminescence properties of the europium folate complexes were studied in phosphate
buffer saline (PBS), RPMI cell culture medium and rat serum (RS) in order to evaluate their
emission behavior and the possible spectral interference from these media prior to the in vitro
evaluation studies (Figure 5). In all the spectra, the typical europium emission bands are clearly
visible between 570 ꢀ 710 nm and the different environments do not affect their shape. The
spectra differ at λ < 550 nm where broad emission bands are present. These bands, having higher
intensity in RS, are likely due to the organic moiety present in both RS and RPMI medium. It is
worth to note that one can get rid of all these bands by performing time gated luminescence
experiments. Indeed, their intensity vanishes if emission is recorded over a 50ꢀ100 ꢁs delayed
3
+
time window after excitation. A comparable behavior has been observed for Eu ⊂1.
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9
0
1
2
3
4
5
6
7
8
9
0
3
+
3+
Figure 5. Emission spectra of Eu ⊂2 30 ꢁM (a) and Eu ⊂3 30 ꢁM (b) in PBS, RPMI cell
culture medium and rat serum excited at 310 nm
Chemical and photophysical stability of the bioconjugated complexes. The in vitro
3+
3+
3+
chemical and photophysical stability of Eu ⊂1, Eu ⊂2 and Eu ⊂3 was determined by HPLC,
monitoring over the time the chemical purity as variation of the concentration of the compounds
after incubation in PBS, RPMI cell culture medium and RS. Samples withdrawn at different
times were analyzed by HPLC, the peaks were integrated and the absolute peak areas of the
complex used to assess its concentrations (µM) by means of the calibration curves. All the
experiments were conducted in duplicate (n = 2). The results expressed as average ± standard
deviation are reported in SI.
The complexes were stable under all investigated conditions. No variation of the chemical
nature of the native compounds was observed after incubation of the complexes in the
investigated media. Indeed, for all the complexes the concentration in each medium remained
3
+
3+
3+
constant over time. The structural integrity of Eu ⊂1, Eu ⊂2 and Eu ⊂3 after incubation in
RPMI cell culture and RS was also confirmed by monitoring their emission spectra at 37°C at
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different time intervals, up to 24 h. Data clearly showed that the europium emissions are
practically unaffected by prolonged treatment at 37 °C, pointing out their suitability for the
3
+
uptake experiments with the cellular lines. As an example, the emission spectra of Eu ⊂2
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collected at different incubation times at 37 °C in RPMI and RS are reported in Figure 6.
3
+
Figure 6. Emission spectra of Eu ⊂2 at different incubation times at 37 °C in RPMI (a) and RS
(b). Irrespective of the medium, the concentration of the complexes is 30 ꢁM
Evaluation of FRα expression and compound cytotoxicity. Since the presence of FRα is a
prerequisite for the uptake of Euꢀlabeled folate, the expression of this receptor was first
evaluated by flow cytometry in a panel of ovarian cancer cell lines, namely SKOVꢀ3, OVCARꢀ3,
IGROVꢀ1 and A2780. Tested cell lines displayed a very variable FRα expression. Indeed,
SKOVꢀ3 was characterized by a strong positivity (>95%), whereas OVCARꢀ3 and IGROVꢀ1
expressed the receptor of interest with an intermediate (about 50ꢀ60%) and low (about 15ꢀ20%)
intensity, respectively. A2780 resulted completely negative (see SI).
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5
6
3+
3+
To test whether the three complexes affected cell growth, cytotoxicity of Eu ⊂1, Eu ⊂2 and
3
+
Eu ⊂3 was carefully investigated for all ovarian cancer cell lines by using PerkinElmer
ATPLite™ test, that estimates the concentration of viable cells on the basis of bioluminescence
generated by the reaction between adenosine triphosphate (ATP) with luciferase and Dꢀluciferin
0
1
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9
0
1
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9
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2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
see SI). Results showed 100% survival for the SKOVꢀ3 cell line up to 30 ꢁM concentration of
3
+
3+
Eu ⊂1 and Eu ⊂2. Cell survival decreased to 80% and 70% at 100 ꢁM and 300 ꢁM
concentrations, respectively. A slight increase in cytotoxicity was observed in the case of
3+
Eu ⊂3, which gave 100% survival up to 10 ꢁM concentration, with percentages decreasing to
95%, 80% and 65% with 30 ꢁM, 100 ꢁM and 300 ꢁM concentrations, respectively. A2780 cells
line that does not express FRs showed 100% survival in all the range of concentrations studied
(
Figure 7). Results of cytotoxicity of the three probes with OVCARꢀ3 and IGROVꢀ1 cell lines
are reported in SI.
Cellular internalization of complexes and competition studies. To assess the cellular
3
+
3+
3+
internalization of Eu ⊂1, Eu ⊂2 and Eu ⊂3 by receptor positive and negative cell lines,
SKOVꢀ3, IGROVꢀ1 and A2780 ovarian cancer cells were incubated for 1, 3, 18, and 24 h with
increasing concentrations of each of the three Eu complexes, in a folic acidꢀdeficient medium. At
each time point, surface bound complexes were removed by washing with glycine buffer. Cells
containing the internalized complex were then lysed with Triton 5% and fluorescence intensity
was measured. Finally, results were plotted against incubation time, according to calibration
curves generated to quantify the amount of internalized complexes. All the measurements were
carried out in triplicate according to the detailed procedure reported in SI. The values of uptake
3+
3+
3+
obtained for Eu ⊂1, Eu ⊂2 and Eu ⊂3 with the SKOVꢀ3 cells are reported in Figure 8.
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5
5
5
5
5
5
5
5
6
3+
3+
Figure 7. In vitro viability of ovarian tumor cell lines in response to Eu ⊂1, Eu ⊂2 and
3
+
TM
Eu ⊂3. A2780 (blue bars) and SKOVꢀ3 (red bars) cell survival was evaluated by ATPlite
assay. Reported values are the mean of three independent experiments, estimated error ± 5%
0
1
2
3
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8
9
0
1
2
3
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0
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3
+
3+
3+
Figure 8. Internalization curves of Eu ⊂1, Eu ⊂2 and Eu ⊂3 by receptorꢀpositive SKOVꢀ3.
Graphs show the amount of complex internalized at different concentrations (30 µM, 1 µM, and
100 nM) and time points (1, 3, 18, and 24 h). Mean values of three independent experiments are
reported
For the SKOVꢀ3 cells, the internalized concentrations strongly increased along with the
increasing concentrations of the probes and the incubation time, being 3.5 nM, 6.0 nM and 1.9
6
6
nM for the 30 ꢁM concentration and the 24h time point, which correspond to 2.1 x 10 , 3.6 x 10
6
3+
3+
3+
and 1.1 x 10 molecules/cell for Eu ⊂1, Eu ⊂2 and Eu ⊂3, respectively. A similar trend,
although with lower values of cellular uptake (e.g., 0.7 nM, 2.0 nM and 0.7 nM at 30 ꢁM and
3
+
3+
3+
24h for Eu ⊂1, Eu ⊂2 and Eu ⊂3, respectively) was obtained with IGROVꢀ1 cells line under
the same experimental conditions (see SI).
These amounts were in the same range as found for other FA derivatives published in
2
4, 35
literature
No internalization was observed for the A2780 cells that do not express FRs, thus
indicating that the internalization of the tested derivatives was receptorꢀmediated. It is interesting
3+
to note that, in both SKOVꢀ3 and IGROVꢀ1 cells, the amount of internalization for Eu ⊂1,
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2
2
2
2
2
2
2
2
2
2
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3
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5
5
5
5
5
6
3+
3+
Eu ⊂2 and Eu ⊂3 were in the same range suggesting that modification on the different
position of the FA structure does not seem significantly affect the recognition by FRα.
To demonstrate that the internalization of the luminescent probes was specifically due to the
0
1
2
3
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9
0
1
2
3
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1
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4
5
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7
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9
0
3
+
presence of FRs, the uptake of Eu ⊂2 by SKOVꢀ3 cells was repeated under the same conditions
reported above, but in the presence of increasing amounts of free FA as competing binding
3
+
substrate. The results reported in Figure 9 clearly show that the uptake of Eu ⊂2 strongly
decreased by the addition of increasing amounts of FA, and was practically absent in the
3
+
presence of FA concentrations about 30 times higher than Eu ⊂2 concentration.
3+
Figure 9. Competitive inhibition of Eu ⊂2 binding to FR on SKOVꢀ3 by FA. Results show the
mean values of three independent experiments
Competition uptake experiments were also carried out in SKOVꢀ3 cells using either FA or
3
+
3
3
Eu ⊂2 as competitive substrates for HꢀFA. In this case, the uptake of HꢀFA, measured in
counts per minute (CPM) with the TopCount NXT apparatus, sharply decreased when cells were
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incubated in the presence of increasing amounts of folic acid or Eu ⊂2. As reported in Figure
3
3+
10, the decrease of HꢀFA binding was the same either with FA or with Eu ⊂2 as competitor
3+
substrates for FR, thus indicating that Eu ⊂2 and free FA display a fully overlapping ability to
10
11
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inhibit the binding of the radioactive folate to SKOVꢀ3.
3
+
3
Figure 10. Comparison of the abilities of either Eu ⊂2 and FA to inhibit HꢀFA binding to
SKOVꢀ3 cells. Results show the mean values of three independent experiments
Finally, to investigate the effects of the uptake process on the chemical integrity of the
3+
complexes, HPLC analysis were conducted on Eu ⊂2 before and after cellular incubation and
internalization, evaluating the chromatographic profile of the compound which remained in the
media after cell incubation, and of the internalized compound recovered from the cytosolic
3+
fraction. Results clearly indicate that Eu ⊂2 remains stable over the time and after cell exposure
and internalization (Figure 11). No evidence of free FA and other species originated by the
3+
hydrolysis of the amide bond in Eu ⊂2 was observed in the postꢀinternalization HPLC profiles,
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even after a 24 h incubation. Indeed, HPLC analysis of the Triton solutions obtained after cell
3
+
lysis and centrifugation revealed the presence of the Eu ⊂2 as unmodified form. Other peaks
present in the UV traces were attributed, by assessment, to the cellular components and/or to the
RPMI cell culture medium.
0
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+
Figure 11. HPLC profiles of SKOVꢀ3 cells lysate, blank (A), Eu ⊂2 at 24 h after internalization
3+
(
B) and Eu ⊂2 in RPMIꢀ1640 cell culture medium, after 24 h of incubation with SKOVꢀ3 cells
(C)
CONCLUSIONS.
3+
3+
In conclusion, we described the preparation of three new luminescent probes Eu ⊂1, Eu ⊂2
3
+
and Eu ⊂3 that are highly specific for cancer cells that overexpress membrane FRs. These
probes are characterized by a luminescent europium complex as the signalling unit, linked in
three different positions to the structure of FA that behaves as the targeting unit. All compounds
were carefully purified and underwent a complete chemical and photophysical characterization
in different media, i.e. water, PBS, RPMI cell culture medium and rat serum. In vitro studies
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have delivered important information regarding the chemical and photophysical stability, as well
as the biochemical properties of these compounds. In particular, these complexes turned out to be
very stable in all conditions investigated, and no biotransformation and no significant interaction
with serum protein occurred. HPLC profiles of the complexes collected after serum incubation
perfectly matched the native compounds. Spectroscopic analysis of the three new luminescent
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3+
3+
probes Eu ⊂1, Eu ⊂2 and Eu ⊂3 were also performed to determine the luminescence
properties of the europium folate complexes before and after media incubation. The collected
data clearly demonstrated that the probes remain intact and that the fluorescence signal was
retained.
3
+
3+
3+
The cytotoxicity of Eu ⊂1, Eu ⊂2 and Eu ⊂3 was tested for SKOVꢀ3, OVCARꢀ3, IGROVꢀ
1
and A2780 human ovarian cancer cells featuring high, medium, low and null FRs expression,
respectively. Results indicate that cell viability was not affected unless extremely high
concentrations of the compounds were reached.
Cellular uptake of these probes was strictly receptorꢀdependent, being critically correlated to
FRs expression. Moreover, it was a function of conjugation site, as observed in SKOVꢀ3 cells
3
+
3+
3+
where the binding activity follows the order Eu ⊂2 > Eu ⊂1 > Eu ⊂3. Notably, the results of
3
competition experiments in the presence of FA or HꢀFA showed a selectivity and specificity of
3
+
Eu ⊂2 probe for the FR comparable to that of FA, thus suggesting that the conjugated moiety
3
+
did not likely affect FA binding properties. Finally, Eu ⊂2 proved to be chemically and
photophysically stable also after the internalization process.
EXPERIMENTAL SECTION.
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Materials and methods. All available chemicals and solvents were purchased from
commercial sources and were used without any further purification. Glycine, hydrochloric acid,
TM
3
Triton Xꢀ100 and folic acid were purchased from SigmaꢀAldrich (St. Louis, MO). Hꢀfolic
acid (14.3 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA). SKOVꢀ3, OVCARꢀ
3, IGROVꢀ1 and A2780 human ovarian cancer cell lines were cultured in RPMI 1640 medium
without folic acid (Life Technologies, Australia) supplemented with 2 mM Lꢀglutamine (Gibco
BRL), 10 mM HEPES (PAA Laboratories, Linz, Austria), 150 U/ml streptomycin (BristolꢀMyers
Squibb Italia, Rome, Italy), 200 U/ml penicillin (Pharmacia and Upjohn, Milan, Italy) and 10%
0
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0
(
v/v) heatꢀinactivated FCS (Gibco BRL, Paisley, UK), hereafter referred to as complete medium.
Thin layer chromatography (TLC) was performed using 0.25 mm silica gel precoated plates Si
60ꢀF254 (Merck, Darmstadt, Germany) visualized by UVꢀ254 light and CAM staining. Column
chromatography was conducted by using silica gel Si 60, 230ꢀ400 mesh, 0.040ꢀ0.063 mm
1
(
Merck). Melting points were determined on a Buchi B450 apparatus and are uncorrected. H
1
3
and C NMR spectra were recorded on a Bruker Avance 400 (400 and 100.6 MHz,
1
respectively), Bruker Fourier 300 or Bruker AMX 300 (recorded at: 300.13 MHz for H; 75.00
13
MHz for C); chemical shifts are indicated in parts per million downfield from SiMe , using the
4
residual proton (CHCl = 7.26 ppm, (CH ) SO = 2.50 ppm, HOD = 4.80 ppm) and carbon
3
3 2
(
CDCl = 77.0 ppm, (CD ) SO = 40.45 ppm) solvent resonances as internal reference. Protons
3 3 2
1
3
1
1
1
13
and carbon assignments were achieved by CꢀAPT, Hꢀ H COSY, and Hꢀ C heteronuclear
correlation experiments. Coupling constants values J are given in Hz. All detailed synthetic
procedures and spectra are reported in the Supporting Information. The chemical purity of the
three optical probes, estimated by HPLC, was ≥ 95%. HPLC analyses were performed using an
®
1
5 cm X 4.6 mm ASCENTIS Cꢀ18 column and, for SemiꢀPreparative HPLC purification, 15
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®
cm X 21.2 mm ASCENTIS Cꢀ18. HPLCꢀgrade acetonitrile was purchased from VWR
International and used without further purification. HPLCꢀaqueous solvent was prepared using
milliꢀQ water (18.2 Mꢂ·cm ionic purity). Optical rotations were measured on a PerkinꢀElmer
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2
41 Polarimeter at 589 nm, using a 10 cm x 5 ml cell and c is in g/100 ml. FTIR spectra were
recorded on an Agilent Cary 630 infrared spectrophotometer and are reported in frequency of
–
1
absorption (cm ). High resolution mass spectra were obtained with an electrospray ionꢀtrap
mass spectrometer ICRꢀFTMS APEX II (Bruker Daltonics) by the Centro Interdipartimentale
Grandi Apparecchiature (C.I.G.A.) of the University of Milano. The assignment of the structure
3+
3+
of the two regioisomers Eu ⊂ 1 and Eu ⊂ 2 was obtained using a LCQ DECA ion trap
instrument (ThermoFisher Scientific, San Josè, CA, USA), operating in positive ion mode. The
3+
3+
entrance capillary temperature was 280 °C and the capillary voltage was 5 kV. Eu ⊂ 1 and Eu
⊂
2 were firstly dissolved in MilliQ water at a concentration of 1 mg/mL and then diluted 1 to
1
00 in water/methanol (50/50, v/v), containing 1% of HCOOH. Water/methanol solutions of
each compounds were introduced into ESI ion source by direct infusion at a flow rate of 8
ꢀL/min. The He pressure inside the trap was kept constant. The pressure directly read by ion
ꢀ5
gauge (in the absence of the N stream) was 2.8·10 Torr. Tandem mass spectrometric (MS/MS)
2
experiments were performed by resonant excitation of the ion of interest through a
supplementary r.f. voltage in the range 30ꢀ35% of its maximum value (5 V peakꢀto peak).
1
51
153
Considering that Eu has two isotopes (Eu and Eu with relative abundance of 47.77% and
52.23% respectively), as clearly shown in Figures S3, the ion chosen for the MS/MS experiments
12
14
1
16
was that at m/z 1109, which is the monoisotopic ones, containing only C , N . H , O and
1
51
Eu .
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3+
3+
Synthesis of Eu ⊂1 and Eu ⊂2 complexes. To a stirred solution of folic acid (220 mg, 0.5
mmol) in 16 mL of anhydrous DMSO and 4 mL of pyridine over KOH was added the 10ꢀ[4ꢀ(3ꢀ
aminopropyloxy)benzoylmethyl]ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriacetic acid europium
0
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complex (Eu ⊂4) (378 mg, 0.55 mmol) and dicyclohexylcarbodiimide (DCC) (258 mg, 1.25
mmol) at room temperature under nitrogen atmosphere. The reaction mixture was magnetically
stirred at room temperature for 16h and the resulting solid was filtered through a glass frit. The
filtrate was slowly dripped into a well stirred Et O (150 mL), the solid precipitate was filtered
2
and washed four time with Et O (10 mL) to remove trace of DMSO and dried under vacuum
2
until constant weight to afford (620 mg) of pale yellow solid. Purification was carried out on a
®
semiꢀpreparative HPLC by using an Ascentis C18 column (5ꢁm, 21.2 x 150 mm) by using as
eluent a mixture of solvents: A = H O (w 0.1% TFA) and solvent B CH CN (w 0.1% TFA)
2
3
starting from 92% A : 8% B to 86% A : 14% B in 20 min, flow rate = 12 ml/min, T=25°C, UV
3
+
detector λ = 310 nm and afforded, after lyophilization 100 mg of Eu ⊂1 (18%) and 210 mg of
3
+
3+
20
Eu ⊂2 (37.8%) (>95% purity by HPLC analysis) as both light yellow solids. Eu ⊂1: [α]
ꢀ
ꢀ
D
ꢀ1
3+
20
D
0
.6 (c 0.4 H O); IR (KBr): 3414.5, 3229.4, 1682.8, 1616.5, 1400.1, 1179.6 cm . Eu ⊂2: [α]
2
ꢀ1
3
.7 (c 0.4 H O); IR (KBr): 3411.3, 3124.0, 1678.0, 1638.4, 1603.5, 1400.2, 1198.7 cm .
2
Photophysical measurements. Absorption spectra in the UVꢀVis were performed using a
doubleꢀbeam CARY5E spectrophotometer with a spectral bandwidth of 1 nm. The luminescence
spectra were recorded at room temperature with a Fluorologꢀ3 (Horiba Jobin Yvon)
spectrofluorimeter equipped with doubleꢀgrating monochromator in both the excitation and
emission sides coupled to a R928P Hamamatsu photomultiplier and a 450 W Xe arc lamp as the
excitation source. The emission spectra were corrected for detection and optical spectral
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response of the spectrofluorimeter through a calibration curve supplied by the manufacturer. The
excitation spectra (PLE) were corrected for the spectral distribution of the lamp intensity using a
photodiode reference detector. Time gated spectra have been acquired with the same instrument
employing a pulsed Xe lamp as source. Absolute photoluminescence quantum yields have been
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®
calculated by corrected emission spectra obtained from an apparatus consisting of a Spectralon
coated integrating sphere accessory (4", Fꢀ3018, Horiba Jobin Yvon), fitted in the fluorimeter
sample chamber. Three independent measurements were carried out on each complex with an
estimated error of ± 20%. The luminescence lifetimes (error ± 10%) in the microsecond–
millisecond scales were measured by a pulsed xenon lamp with variable repetition rate and
elaborated with standard software fitting procedures.
Stability studies in PBS, RPMI and RS. HPLC was used to evaluate the chemical purity and
the stability of the complexes was measured on a Beckman System Gold instrument equipped
with a programmable solvent Model 126 and a scanning detector Module 166. Analyses were
carried out on a RP Symmetry300 C4 Guard Column (5ꢁm 3.9 x 20 mm) and a RP
Symmetry300 C4 column (5ꢀm 4,6 x 150 mm). Solvents: A = H O (w 0.1% TFA); B CH CN (w
2
3
0
.1% TFA). Gradient: 0ꢀ8 min %B=0 (isocratic), 8ꢀ23 min %B=20 (gradient), 23ꢀ33 min %B=65
(gradient), 33ꢀ38 min %B=65 (isocratic), 38ꢀ39 min %B=0 (gradient), 39ꢀ40 min %B=0
3+
(
isocratic). Flow rate of 1 mL/min. UV Detector λ = 305 nm. Retention times: Eu ⊂1 Rt =
3
+
3+
21.14±0.11; Eu ⊂2 Rt = 22.88 ± 0.18; Eu ⊂3 Rt = 23.79 ± 0.06.
Evaluation procedures for the in vitro stability. For each compound, a stock solution was
3
+
3+
3+
prepared dissolving Eu ⊂1, Eu ⊂2 or Eu ⊂3 in DMSO (1 mM) and used to achieve a final
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