Quantum Dot Based Luminescent Nanoprobes for Sigma-2 Receptor Imaging

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00825

The increasing importance of sigma-2 receptor as target for the diagnosis and therapy of tumors paves the way for the development of innovative optically traceable fluorescent probes as tumor cell contrast and therapeutic agents. Here, a novel hybrid organic-inorganic nanostructure is developed by combining the superior fluorescent properties of inorganic quantum dots (QDs), coated with a hydrophilic silica shell (QD@SiO₂ NPs), the versatility of the silica shell, and the high selectivity for sigma-2 receptor of the two synthetic ligands, namely, the 6-[(6-aminohexyl)oxy]-2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-dihydroisoquinolin-1(2H)-one (MLP66) and 6-[1-[3-(4-cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-yloxy]hexylamine (TA6). The proposed nanostructures represent a challenging alternative to all previously studied organic small fluorescent molecules, based on the same sigma-2 receptor affinity moieties. Flow cytometry and confocal fluorescence microscopy experiments, respectively, on fixed and living cancerous MCF7 cells, which overexpress the sigma-2 receptor, prove the ability of functionalized (QD@SiO₂-TA6 and QD@SiO₂-MLP66) NPs to be internalized and demonstrate their affinity to the sigma-2 receptor, ultimately validating the targeting properties conveyed to the NPs by sigma-2 ligand conjugation. The presented QD-based nanoprobes possess a great potential as in vitro selective sigma-2 receptor imaging agent and, consequently, could provide a significant impact to future theranostic applications.

Full text of DOI:10.1021/acs.molpharmaceut.7b00825

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Article
Quantum Dot Based Luminescent Nanoprobes for Sigma-2 Receptor Imaging
Maria Laura Pati, Elisabetta Fanizza, Sonja Hager, Diana Groza, Petra Heffeter, Amelita Grazia
Laurenza, Valentino Laquintana, Maria Lucia Curri, Nicoletta Depalo, Carmen Abate, and Nunzio Denora
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00825 • Publication Date (Web): 11 Dec 2017
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Quantum Dot Based Luminescent Nanoprobes for Sigma-2 Receptor
Imaging
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Maria Laura Pati,†,a Elisabetta Fanizza,‡,§,a Sonja Hager,# Diana Groza,# Petra Heffeter,#
Amelita Grazia Laurenza,‡ Valentino Laquintana, † Maria Lucia Curri,§ Nicoletta Depalo,§,*
Carmen Abate†,* and Nunzio Denora†
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† Dipartimento di FarmaciaꢀScienze del Farmaco, Università degli Studi di Bari Aldo Moro,
Via Orabona 4, Iꢀ70125 Bari, Italy.
§Istituto per i Processi ChimicoꢀFisici ꢀ IPCF ꢀ SS Bari, Consiglio Nazionale delle Ricerche,
c/o Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro Via Orabona 4, 70125
Bari, Italy
‡Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via Orabona 4, Iꢀ70125
Bari, Italy
#Department of Medicine I, Institute of Cancer Research and Comprehensive Cancer Center
of the Medical University, Medical University of Vienna, Borschkegasse 8a, Aꢀ1090 Wien,
Austria
*Corresponding Author: n.depalo@ba.ipcf.cnr.it Istituto per i Processi ChimicoꢀFisici ꢀ IPCF
ꢀ SS Bari, Consiglio Nazionale delle Ricerche, c/o Dipartimento di Chimica, Università degli
Studi di Bari Aldo Moro Via Orabona 4, 70125 Bari, Italy
carmen.abate@uniba.it Dipartimento di FarmaciaꢀScienze del Farmaco, Università degli Studi
di Bari Aldo Moro, Via Orabona 4, Iꢀ70125 Bari, Italy
Abstract. The increasing importance of sigmaꢀ2 receptor as target for the diagnosis and
therapy of tumours paves the way for the development of innovative optically traceable
fluorescent probes as tumour cell contrast and therapeutic agents. Here, a novel hybrid
organicꢀinorganic nanostructure is developed by combining the superior fluorescent properties
of inorganic quantum dots (QDs), coated with a hydrophilic silica shell (QD@SiO2 NPs), the
versatility of the silica shell and the high selectivity for sigmaꢀ2 receptors of the two synthetic
ligands, namely the 6ꢀ[(6ꢀaminohexyl)oxy]ꢀ2ꢀ(3ꢀ(6,7ꢀdimethoxyꢀ3,4ꢀdihydroisoquinolinꢀ
2(1H)ꢀyl)propyl)ꢀ3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀone
(MLP66)
and
6ꢀ[1ꢀ[3ꢀ(4ꢀ
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cyclohexylpiperazinꢀ1ꢀyl)propyl]ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ5ꢀyloxy]hexylamine (TA6).
Therefore, the proposed nanostructures represent a challenging alternative to all previously
studied organic small fluorescent molecules, based on the same sigmaꢀ2 receptor affinity
moieties. Flow cytometry and confocal fluorescence microscopy experiments, respectively,
on fixed and living cancerous MCF7 cells, which overexpress the sigmaꢀ2 receptors, prove
the ability of functionalized (QD@SiO2ꢀTA6 and QD@SiO2ꢀMLP66) NPs to be internalized
and demonstrate their affinity to the sigmaꢀ2 receptors, ultimately validating the targeting
properties conveyed to the NPs by sigmaꢀ2 ligand conjugation. The presented QDꢀbased
nanoprobes possess a great potential as in vitro selective sigmaꢀ2 receptor imaging agent and,
consequently, could provide a significant impact to future theranostic applications.
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Keywords: hybrid nanostructure, fluorescent bioimaging, quantum dots, sigmaꢀ2 receptor
1. Introduction. In spite of the overall decrease of mortality rate in the past 20 years,1 cancer
is still the second leading cause of death, as some tumours are still characterized by an
increasing incidence (e.g. liver, pancreas, thyroid, leukemia tumour), and expected to overtake
the prevalence of cardiovascular diseases within the next years. One of the major open
challenges in treating cancers is to increase the tumour specificity of chemotherapeutic agents
in order to reduce the often severe side effects causing damage to normal tissues and organs.2
This goal can be accomplished by developing an efficient targeting delivery nanosystem, able
to direct selective binding to cancer cells. Indeed, clinical nanotechnology based strategies
have proven that nanoparticle based delivery platforms can effectively enhance therapeutic
efficacy, improve targeted delivery to tumours and active cellular uptake while
simultaneously reducing side effects.3ꢀ6 In this scenario, several inorganic colloidal
nanoparticles (NPs), and nanocrystals (NCs), including plasmonic gold NPs,7ꢀ8 luminescent
quantum dots (QDs),9ꢀ10 superparamagnetic iron oxide NPs11ꢀ12 as well as
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nanoheterostructures13 have demonstrated a vast potential as targeted therapeutic and/or
diagnostic nanovectors.14ꢀ16 Indeed, their high surface to volume ratio, their peculiar size
dependent physicochemical properties, such as optical or magnetic characteristics, and their
ability to be surface engineered with a large variety of ligands to enhance their affinity
towards target sites point out the enormous capability of such colloidal nanostructures.17ꢀ22
Among the synthetic ligands that can be conjugated to NP surface for an active targeting of
tumour tissues, small molecules offer a number of advantages, such as high degree of purity,
scalability of production, lack of immunogenicity as well as elevated affinity and selectivity
for the receptors.23 Receptors overexpressed in tumour cells represent promising targets for
binding cancer cells. In this perspective, two subtypes of sigma receptors (namely subtypeꢀ1,
sigmaꢀ1 receptor and subtypeꢀ2, sigmaꢀ2 receptor), overexpressed in cancer cells from animal
and human origins, are gaining increasing attention as tumour targets. While the subtypeꢀ1 of
sigma receptors has been already cloned and crystallized,24 the subtypeꢀ2 has still to be clearly
identified. In fact, sigmaꢀ2 has been first proposed as a histone protein,25 and later as the
progesterone receptor membrane component 1 (PGRMC1) protein complex,26 with strong
evidence supporting the nonꢀidentity of the sigmaꢀ2 protein with the PGRMC1.27ꢀ29 Only
recently, this receptor has been proposed as the endoplasmic reticulum (ER)ꢀresident
membrane protein TMEM97.30 Despite the still unclear identification, the interest in the
sigmaꢀ2 receptor research is recently increasing as an exceptional candidate tumour imaging
target, since it is expressed about 10ꢀfold more in proliferating tumour cells than in quiescent
tumour cells. In addition, ligand binding to this receptor can result in tumour cell death via
both apoptotic and nonꢀapoptotic mechanisms.31 Therefore, sigmaꢀ2 proteins represent
promising targets for the therapy and diagnosis of tumours, with significant preliminary
results obtained from cell culture and from preclinical models of aggressive tumours such as
the pancreas carcinoma.32ꢀ34 In addition, clinical phase I studies for the diagnosis of different
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tumours are already ongoing.35ꢀ36 In literature, few reports on the use of sigmaꢀ2 receptors
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ligands conjugated to inorganic NPs for their targeted delivery into cancer cells have been
presented.37ꢀ39 A new class of conjugates composed of gold nanocages and a ligand specific to
sigmaꢀ2 receptors (SV119) have been proposed37 as a multifunctional and theranostic
platform for cancer targeting. Furthermore, anisamide ligandꢀconjugated gold nanospheres
have been investigated for the targeting of sigma receptors, although the preferential binding
of anisamide to either subtypes has not been demonstrated.40ꢀ41 On this basis and in order to
support sigmaꢀ2 receptors for selective cancer targeting, in this work, novel sigmaꢀ2ꢀ
directioning luminescent nanostructures have been prepared by conjugating luminescent
semiconductor QDs, with two different specific sigmaꢀ2 receptor ligands. Luminescent QDs
have been selected as fluorescent contrast agents due to their superior optical properties with
respect to conventional organic fluorophores, including stability to photobleaching, long
fluorescence (PL) lifetime, enabling longꢀterm and sensitive imaging and wide absorption
spectrum.17,18,42ꢀ43 In particular, amineꢀfunctionalized luminescent silicaꢀcoated (CdSe)ZnS
QDs, namely (QD@SiO2ꢀNH2 NPs) have been successfully conjugated to two structurally
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different
small
molecules,
the
6ꢀ[(6ꢀaminohexyl)oxy]ꢀ2ꢀ(3ꢀ(6,7ꢀdimethoxyꢀ3,4ꢀ
dihydroisoquinolinꢀ2(1H)ꢀyl)propyl)ꢀ3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀone (MLP66) and 6ꢀ[1ꢀ
[3ꢀ(4ꢀcyclohexylpiperazinꢀ1ꢀyl)propyl]ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ5ꢀyloxy]hexylamine
(TA6).44 The ligand structures have been purposely designed on the bases of two of the
highest affinity and selectivity sigmaꢀ2 receptor ligands previously reported (the 2ꢀ(3ꢀ(6,7ꢀ
dimethoxyꢀ3,4ꢀdihydroisoquinolinꢀ2(1H)ꢀyl)propyl)ꢀ6ꢀmethoxyꢀ3,4ꢀdihydroisoquinolinꢀ
1(2H)ꢀone
(1)45
for
the
MLP66
and
1ꢀcyclohexylꢀ4ꢀ[3ꢀ(5ꢀmethoxyꢀ1,2,3,4ꢀ
tetrahydronaphthalenꢀ1ꢀyl)ꢀnꢀpropyl]piperazine (PB28)46 for the TA6, respectively). This
synthetic strategy has allowed to fabricate two different types of single nanoꢀobjects, each
combining the high affinity of the specific ligand towards sigmaꢀ2ꢀreceptors and the peculiar
emission properties of the QD@SiO2 NPs. The obtained highly luminescent nanoplatforms
are the first examples of sigmaꢀ2ꢀtargeted QDꢀbased nanostructures that have been obtained
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and represent powerful optically traceable tools to investigate sigmaꢀ2 receptors at the
subcellular level. Original insights on the sigmaꢀ2 receptor physiological role and its
mechanism of action could thus be obtained in future by taking advantage of the superior
fluorescent properties of QD@SiO2 while tracing the path to the targeted delivery of deathꢀ
inducing payloads to sigmaꢀ2ꢀoverexpressing cancer cells. In this perspective, the diagnostic
potential of the proposed luminescent targeted nanostructures could be extended to cancer
therapy, or to adjuvant anticancer treatment.
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2. Materials and Methods.
2.1 Chemistry. Column chromatography was performed with 60 Å pore size silica gel
as the stationary phase (1:30 w/w, 63ꢀ200 ꢁm particle size from ICN). Melting points
1
were determined in open capillaries on a Gallenkamp electrothermal apparatus. H
NMR (300 MHz) spectra were recorded on a Mercury Varian spectrometer. CDCl3 was
1
used as solvent to record H NMR unless otherwise stated. The following data were
reported: chemical shift (d) in ppm, multiplicity (s = singlet, d = doublet, t = triplet, m
= multiplet), integration and coupling constant(s) in Hertz. Recording of mass spectra
was done on an Agilent 6890ꢀ5973 MSD gas chromatograph/mass spectrometer and on
an Agilent 1100 series LCꢀMSD trap system VL mass spectrometer; only significant
m/z peaks, with their percentage of relative intensity in parentheses, are reported.
Chemicals were from Aldrich, Alfa Aesar or TCI and were used without any further
purification. Details of the synthesis for the sigmaꢀ2 receptor ligands are reported in
the Supplementary Information file.
2.1.1 6-(2,5-Dimethylpyrrol-1-yl)hexan-1-ol (
(0.8 g, 8 mmol) in anhydrous toluene (20 mL), acetonyl acetone (0.93 g, 8.9 mmol)
and a catalytic amount of ꢀtoluensulfonic acid were added and the mixture was
2). To a solution of 6ꢀaminoꢀ1ꢀhexanol
p
refluxed for 6 h during which water was removed through a Dean Stark apparatus. The
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solvent was then removed under reduced pressure and the red dense oil obtained was
purified by column chromatography (CH2Cl2/AcOEt, 8:2) to provide the title
compound as an orange oil (82% yield); GC/MS m/z: 301 (M+, 195), 108 (100).
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2.1.2. 6-(2,5-Dimethylpyrrol-1-yl)hexyl methansulfonate (
3). To a solution of
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compound (17.77 mmol, 2.88 g) in dry CH2Cl2 (30 mL) cooled in a dry ice bath,
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Et3N (37 mmol, 5.14 mL) and methansulfonyl chloride (17.72 mmol, 1.36 mL) were
added dropwise and the solution was stirred at the same temperature for 1 h. Water was
then added to the reaction (15 mL) and the mixture was extracted with CH2Cl2 (3 × 20
mL), the organic phases collected and dried (Na2SO4). The crude obtained was purified
by column chromatography (AcOEt/nꢀHexane; 1:1) affording the title compound as a
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brown oil (70% yield); H NMR (300 MHz) δ 1.35ꢀ1.85 [m, 8H, (CH2)4], 2.20 [s, 6H,
(ArCH3)2], 3.00 (s, 3H, SO2CH3), 3.72 (t, 2H,
J = 7.7 Hz, CH2N), 4.22 (t, 2H, J = 6.5
Hz, CH2N), 5.70 (s, 2H, aromatic); LCꢀMS (ESI+) m/z: 296 [M+Na]+; LCꢀMSꢀMS
296: 178, 108.
2.1.3. 6-Hydroxy-3,4-dihydro-isoquinolin-(2H)-1-one (4). CCl3COOH (4.0 g) and 5ꢀ
hydroxyindanꢀ1ꢀone (2.70 mmol, 4.0 g) were heated up to 90 °C until the mixture melt.
NaN3 (0.35, 5.4 mmol) was added and the mixture was warmed at 90 °C for 4 h. The
reaction vessel was then cooled, added with ice and neutralized with KHCO3 (saturated
solution). The aqueous phase was extracted with AcOEt (3 × 10 mL) and the organic
phases were collected and dried (Na2SO4) to provide a crude solid that was purified by
column chromatography (CH2Cl2/AcOEt, 1:1), to provide the title compound as a
white solid (42% yield) ); 1H NMR (CD3OD) δ 2.88 (t, 2H,
J = 6.6 Hz ArCH2), 3.45 (t,
2H,
J = 6.7 Hz NHCH2), 6.60ꢀ6.70 (m, 2H, aromatic), 7.75ꢀ7.85 (m, 1H, aromatic);
LCꢀMS (ESI+) m/z: 296 [M+Na]+; LCꢀMSꢀMS 296: 178, 108.
2.1.4. 6-Benzyloxy-2-(3-chloropropyl)-3,4-dihydro-isoquinolin-(2H)-1-one (6). To a
suspension of NaH (4.35 mmol, 0.104 g) in anhydrous DMF (5 mL) cooled on an ice
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bath, 6ꢀbenzyloxyꢀ3,4ꢀdihydroꢀisoquinolinꢀ(2H)ꢀ1ꢀone 5 (1.74 mmol, 0.44 g) dissolved
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in the same solvent (10 mL) was added dropwise. The mixture was stirred at the same
temperature for 15 min and afterwards added with 1ꢀBromoꢀ3ꢀchloropropane (1.91
mmol, 0.27 mL), and left under stirring at room temperature for 1h. Excess of NaH
was then quenched with water upon cooling, and the solvent was evaporated under
reduced pressure. The crude residue was taken up with water and extracted with
CH2Cl2 (3 × 20 mL), the organic phases were collected and dried (Na2SO4) to provide
a crude mixture that was purified by column chromatography (CH2Cl2/AcOEt, 8:2), to
provide the title compound as a white solid (78% yield); GC/MS m/z: 329 (M+, 195),
294 (30), 91 (100).
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2.1.5.
2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-
). To a solution of (1.35 mmol, 0.45 g)
dihydroisoquinolin-6- benzyloxy-1(2H)-one (
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in DMF (20 mL), 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline (1.49 mmol, 0.29 g)
and K2CO3 (1.50 mmol, 0.21 g) were added and the mixture was warmed under stirring
at 100 °C for 6 h and at room temperature overnight. After cooling to room
temperature, the solvent was evaporated under vacuum and the residue was treated
with H2O and extracted with CH2Cl2 (3 × 15 mL). The collected organic layers, were
dried (Na2SO4) and concentrated under reduced pressure to provide a crude residue
which was purified by column chromatography with CH2Cl2/MeOH (95:5) as eluent,
to provide the title compound as a dense yellow oil (40% yield); LCꢀMS (ESI+) m/z
:
509 [M+Na]+; LCꢀMSꢀMS 509: 403.
2.1.6.
2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-
). A solution of (0.49 mmol, 0.24 g) in
dihydroisoquinolin-6- hydroxy-1(2H)-one (
8
7
MeOH (7 mL) was added with Pd 10% on activated charcoal and stirred under H2 (4
atm) overnight. The mixture was filtrated through Celite to remove the catalyst and the
filtrate was evaporated under vacuum to provide a brown oil residue which was
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purified by column chromatography with CH2Cl2/MeOH (9:1) as eluent, to provide the
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title compound as a waxy solid (55% yield); H NMR δ 1.85ꢀ2.00 (m, 2H,
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CH2CH2CH2), 2.49 (t, 2H,
4H, ArCH2), 3.29 (t, 2H,
D2O exchanged), 3.67 (s, 2H, NCH2Ar), 3.85 (s, 6H, OCH3), 6.32 (d, 1H,
aromatic), 6.51 (s, 1H, aromatic), 6.56 (s, 1H, aromatic), 6.71 (dd, 1H, = 8.5 Hz, J’
2.4 Hz, aromatic), 7.88 (d, 1H, = 8.5 Hz, aromatic).
2.1.7. 2-[3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl]-6-{[6-(2,5-
dimethyl-1H-pyrrol-1-yl)hexyl]oxy}-3,4-dihydroisoquinolin-1(2H)-one To
solution of the intermediate compound (0.16 mmol, 0.066 g) in CH3CN (10 mL),
mesyl derivative (0.2 mmol, 0.055 g) and K2CO3 (0.4 mmol, 0.055 g) were added
J
= 6.6 Hz, NCH2), 2.65ꢀ2.75 (m, 2H, NCH2), 2.85ꢀ2.95 (m,
= 6.6 Hz, CONCH2), 3.56ꢀ3.60 (m, 3H, CONCH2 and OH,
= 2.4 Hz,
J
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J
J
=
J
(
9
).
a
8
3
and the mixture was refluxed under stirring overnight. The solvent was evaporated
under vacuum and the residue was treated with H2O and extracted with AcOEt (3 × 10
mL). The collected organic layers were dried (Na2SO4) and concentrated under
reduced pressure to provide a dense yellow oil, which was purified by column
chromatography with CH2Cl2/MeOH (98:2) as eluent. The title compound was
obtained as a yellow oil (65% yield); LCꢀMS (ESI+) m/z: 596 [M+Na]+.
2.1.8.
yl)propyl)-3,4-dihydroisoquinolin-1(2H)-one
0.04 g) was added to a solution of the intermediate
6-[(6-Aminohexyl)oxy]-2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
MLP66). NH2OH·HCl (0.57 mmol,
(0.11 mmol, 0.07 g) in EtOH/H2O
(
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(5 mL, 1/1) and the mixture was refluxed under stirring for 24 h. After cooling, NaOH
(1.15 mmol, 0.046 g) was added to the mixture, which was refluxed under stirring for
further 24 h. The solution was then concentrated under reduced pressure and the
residue was taken up with H2O (3 mL) and extracted with AcOEt (3 × 5 mL). The
collected organic layers were dried (Na2SO4) and concentrated under reduced pressure
to provide a dense yellow oil, which was purified as oxalate salt and recrystallized
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from MeOH/Et2O (60% yield); H NMR δ 1.35ꢀ1.90 (m, 10H, CH2(CH2)4 and
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CH2CH2CH2), 1.95ꢀ2.00 (m, 2H, NCH2), 2.58ꢀ2.98 (m, 8H, 2 NCH2, 2 ArCH2), 3.55ꢀ
3.65 (m, 6H, CONCH2 and ArCH2N), 3.83 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.95ꢀ
4.02 (m, 2H, OCH2), 6.55 (s, 1H, aromatic), 6.60 (s, 1H, aromatic), 6.65ꢀ6.83 (m, 2H,
aromatic), 7.92ꢀ8.05 (m, 1H, aromatic); LCꢀMS (ESI+) m/z: 518 [M+Na]+; 496
[M+H]+; LCꢀMSꢀMS 496: 303, 204, 176.
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2.2 Synthesis and characterization of silica-coated QDs
2.2.1 Materials. Cadmium oxide (99.5% powder, CdO), Trioctylphosphine oxide
(99%, TOPO), tꢀButylphosphonic acid (98%, tBuPOH), Hexadecylamine (technical
grade 90%, HDA, Fluka), Tributylphosphine (97%, TBP), Selenium (99.99% powder,
Se), Sulfur (99.9999% powder, S), Trioctylphosphine (90%, TOP), Diethylzinc
solution (1 M in heptanes, Et2Zn), Hexamethyldisilathiane (HMST) orthosilicate (98%
d = 0.934 g/mL, TEOS), aqueous ammonia solution (d = 0.900 g/mL, NH4OH), 5ꢀ
polyoxyethylene
nonylphenylether
(Igepal
COꢀ520,
Mn=441),
3ꢀ
Aminopropyl)triethoxysilane (97%, APS), Ninhydrin, 2,6ꢀlutidine (98%)
bis(sulfosuccinimidyl)suberate (≥95%, BS3), boric acid and dimethyl sulfoxide
(DMSO) were all purchased from Sigma Aldrich.
2.2.2 Synthesis of QD@SiO2 NPs functionalized with sigma-2 receptor ligands.
CdSe@ZnS QDs with emission centered at 597 nm (d = 4 nm and relative standard
deviation σ% = 20%, for absorption and emission spectra and transmission electron
micrograph, see Supporting Information Figure S1), synthesized as previously
[47,48]
reported,
was coated with a silica shell by means of waterꢀinꢀoil microemulsion
approach (6 mL of ciclohexane, 50 µL of CdSe@ZnS QD solution at 1.8 • 10ꢀ4 M, 350
µL IGEPALꢀCO520, 200 µL ammonia solution 28%, 50 µL TEOS). The QD@SiO2
NPs were dispersed in 2 mL of ethanol. Amine groups were grafted on NP surface by
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condensation reaction carried out at high pH condition between siloxane groups of the
APS and silanol groups at the silica surface, (0.7 mmol of APS added to 400 µL of
QD@SiO2 NPs diluted to 2 mL ethanol), and the degree of functionalization was
measured by a ninhidrine assay, able to titrate the amine groups at the surface (1
mg/mL of QD@SiO2ꢀNH2 with [NH2] of nearly 4·10ꢀ3M).13, 47 TA6 was crosslinked at
the QD@SiO2ꢀNH2 NPs by using BS3 as cross coupling agent in 2 mL of
DMSO/borate buffer 50 mM at pH 8.5 (nearly 2:1). In particular, 500 ꢁL of
QD@SiO2ꢀNH2 NPs in ethanol (2·10ꢀ6mol of NH2 groups) were centrifuged,
redispersed in 500 ꢁL of DMSO/borate buffer (1:1) and mixed with an equimolar
solution of TA6 in 1 mL of DMSO/borate buffer (4:1). Finally, an excess of BS3, 2·10ꢀ
5 mol in 500 ꢁL of DMSO/borate buffer (1:1) was added and the reaction mixture
stirred for 2 h before being quenched by addiction of 10 ꢁL of TRIS HCl. QD@SiO2ꢀ
TA6 NPs were finally collected by repeated cycles of centrifugation at 7000×g and
redispersion in DMSO and finally suspended in DMSO for the spectroscopic
characterization and in H2O for hydrodynamic diameter, size distribution colloidal
stability and in vitro experiments. Functionalization of QD@SiO2ꢀNH2 NPs with
MLP66 was carried out by using the same crosslinking reactions protocol.
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The TA6 and MLP66 content in QD@SiO2–TA6 and QD@SiO2–MLP66 NPs
respectively was determined by spectrofluorometric characterization prior to
construction of a calibration curve. In particular, fluorescent spectra of each ligand
(λex= 300 nm) at different concentration were recorded. The calibration curve was
obtained by plotting the integrated area of the emission band in the wavelength range
between 330 and 500 nm and 330ꢀ580 nm, for TA6 and MLP66, respectively, versus
ligand concentration.
2.3 Transmission Electron Microscopy (TEM). TEM images were acquired by using a
JEOL 100, operating at 100 kV and a Quemesa Olympus CCD 11 Mp Camera.
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Starting from a NP suspension in ethanol, the samples were deposed on 300 mesh
amorphous carbonꢀcoated Cu grid by dipping and evaporation of the solvent. Size
statistical analysis (average NP size and relative standard deviation (σ%) for each NP
sample) was performed by means of a freeware Image J analysis program, to provide
information on the NP size distribution.
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2.4 Spectroscopic characterization. For the spectroscopy characterization, performed at
room temperature the following equipments were used: UVꢀVis absorbance spectra were
recorded by a Cary Varian 5000 UVꢀvisibleꢀNIR, spectrophotometerfluorescent
spectra were recorded by spectrofluorimeter Fluorolog 3 (HORIBA JobinꢀYvon)
equipped with double chromator reticles in excitation and emission and absolute PL
QY were measured dispersing the NP samples in DMSO and using the
spectrofluorimeter equipped with an integration sphere. Infrared spectra in Fourier
transform infrared (FTIR) mode with a spectral resolution of 4 cm−1 were recorded
with a PerkinꢀElmer Spectrum One FTIR spectrometer equipped with a DTGS
(deuterated triglycine sulphate) detector.
2.5 Particle size, size distribution and surface charge. Hydrodynamic diameter (size)
and size distribution (reported in terms of numbers and polydispersity index (PDI),
respectively) for the colloidal nanosystem samples were determined at each
functionalization step by means of dynamic light scattering (DLS). The investigation
was perfomed by diluting the NP aqueous solution (borate buffer, 50 mM, pH 8.5) in
demineralized water. Similarly, NP aqueous solutions (borate buffer, 50 mM, pH 8.5)
were diluted in KCl aqueous solution (1 mM) for ζꢀpotential measurements, carried out
by using a laser doppler velocimetry (LDV) performed in triplicates. All these analysis
were carried out using a Zetasizer Nano ZS, Malvern Instruments Ltd., Worcestershire,
UK (DTS 5.00).
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2.6 Cell biology.
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2.6.1 Materials. Cell culture reagents were freshly prepared in the core facility of the
Institute of Cancer Research by using components purchased from Sigma Aldrich. The
human breast adenocarcinoma cell line MCF7 was obtained from Interlab Cell Line
Collection (ICLC, Genoa) and was grown in DMEM high glucose supplemented with
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10% fetal bovine serum in a humidified incubator at 37 °C with a 5% CO2 atmosphere
.
2.6.2 Cytotoxicity tests in cancer cell lines (MTT assay). To determine the impact of
the new nanocarriers on cell viability of MCF7 cells, 105 cells/mL were plated on 96ꢀ
well plates (100 µL/well) and allowed to recover for 24 h. Then, cells were exposed to
the test drugs with the different concentrations (from 0.05 to 0.0001 mg/mL) for 72 h.
Anticancer activity was measured by the 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT)ꢀbased vitality assay (EZ4U; Biomedica, Vienna,
Austria) following the manufacturer’s recommendations. Cytotoxicity was calculated
using the Graph Pad Prism software (using a pointꢀtoꢀpoint function) and was
expressed as EC50 values calculated from full dose–response curves (drug
concentrations inducing a 50% reduction of cell number in comparison to untreated
control cells cultured in parallel).
2.6.3 Flow cytometry analysis. Cells were incubated with QD@SiO2–NH2,
QD@SiO2–TA6 or QD@SiO2–MLP66 NPs (0.010 mg/mL) for different time point (3
h, 5 h and 24 h) at 37 °C. At the end of the incubation period, cells were washed twice
with phosphateꢀbuffered saline (PBS), detached with Trypsin/EDTA (freshly prepared
in the core facility of the Institute of Cancer Research, by using components purchased
from Sigma Aldrich) for 10 min at 37°C, centrifuged at 1200 rpm for 5 min and reꢀ
suspended in 250 µL of PBS. The fluorescence was recorded using a FACS Calibur
(Becton Dickinson, Palo Alto, CA) with a 585 nm bandꢀpass filter. For each analysis,
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10,000 events were collected and analyzed by ModeFit software (Becton Dickinson
and Company, New York, USA).
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2.6.4 Confocal microscopy analysis. For experiments on live cells, 105 cells were
grown on sterile µꢀSlide 8 Well Glass Bottom (Ibidi) and treated with QD@SiO2–NH2,
QD@SiO2–TA6 or QD@SiO2–TA6 NPs for 24 h followed by NO1 or F412 treatment
for 45 min, and then live cells were imaged with a Zeiss LSM 700 Olympus (Carl
Zeiss AG, Oberkochen, Germany). For each experimental point, a minimum of three
microscopic fields was examined. For experiments on fixed cells, 105 cells were grown
on sterile microscope slides with removable chambers, rinsed and fixed with 4% w/v
paraformaldehyde for 15 min, then rinsed and permeabilized with 0.5% tritonꢀX 100
for 20 min. The samples were then washed with PBS and stained with QD@SiO2–NH2
or QD@SiO2–TA6 or QD@SiO2–MLP66 NPs (0.010 mg/mL) for 24 h. After washing
with PBS, samples were incubated with DAPI (Sigma Aldrich) for 10 min and then
washed. The coverslips were mounted with 6 ꢁL of Vectashield Mounting Medium
and examined with a Zeiss LSM 700 Olympus (Carl Zeiss AG, Oberkochen,
Germany). For each experimental point, a minimum of three microscopic fields was
examined.
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2.6.5 Live cell microscopy analysis. 105 cells were grown on sterile µꢀSlide 8 Well Glass
Bottom (Ibidi) and treated with QD@SiO2–NH2, QD@SiO2–TA6 or QD@SiO2–
MLP66 NPs (0.010 mg/mL) alone or in combination with F412 (10 µM). QD@SiO2
NPs were added 2 h before F412. The imaging started after the addition of the greenꢀ
emitting compound using Nikon eclipse Tiꢀe fluorescence microscope with a sCMOS
pco.edge camera (Nikon Instruments Europe B.V., Amsterdam, Netherlands).
3. Results and Discussion
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Silicaꢀcoated luminescent QDs were used as robust inorganic platforms to conjugate two
structurally different sigmaꢀ2 receptor ligands, namely TA6 and MLP66, here proposed as
sigmaꢀ2 receptor ligands, in order to generate a luminescent nanoparticleꢀbased optically
traceable tool for sigmaꢀ2 receptor targeting. A scheme depicting the steps leading to silicaꢀ
coated luminescent QD conjugated with TA6 or MLP66 ligands is reported in Chart 1.
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Chart 1. Scheme of the synthetic path for silicaꢀcoated QDs functionalization with sigmaꢀ2
receptor ligands. QDs are coated with a silica shell by means of a microemulsion approach
(Step 1), functionalized with amine groups, through
a
reaction with 3ꢀ
(aminopropyl)trimethoxysilane in aqueous ammonia solution (Step 2), and further conjugated
to sigmaꢀ2ꢀreceptor ligands (TA6 or MLP66) by cross coupling reaction mediated by the
bis(sulfosuccinimidyl)suberate (Step 3). See experimental section for details.
The proposed hybrid nanostructures aim at ingeniously combining the optical properties of
QDs with the high affinity and selectivity for sigmaꢀ2ꢀreceptors of the two purposely designed
ligands, in order to fabricate efficient sigmaꢀ2 targeting luminescent probes. Although
promising fluorescent molecular systems based on the 1ꢀ and PB28ꢀstructure sigmaꢀ2 affinity
agonists (see Figure 1) have been already proposed as optical probe targeting the sigmaꢀ2
49ꢀ50
receptor,
they suffer from low fluorescence quantum yield (QY) (below 0.6, with higher
value reached in nonꢀpolar solvent or when ligand bound hydrophobic sites). Therefore, the
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replacement of the fluorescent tag with inorganic luminescent QDs will potentially bring
advantages by exploiting the much more stable photophysical properties of the QDs, i.e.
narrow and brighter emission, high QY, reduced photobleaching effect and longer
fluorescence lifetime, which may contribute to improve sensitivity, signal to noise of the
hybrid fluorescent probe for in vitro imaging. Encapsulation of the QDs inside the silica
matrix represents a wide standing procedure to make the hydrophobic QD dispersable in
aqueous media and also to overcome their limitations for application in the biological field,
mainly the effect of surface modification on QD photophysical properties and the release of
toxic heavy metal ions. The hydrophilicity of the silica matrix, together with its inertness,
good level of biocompatibility and easy of functionalization, will provide a nanostructure with
relevant colloidal stability in aqueous media and high density of covalently bound ligands,
that, in principle offers multiple and simultaneous interactions with cell surface, thus resulting
highly active and still selective for the optically traceable targeting of sigmaꢀ2ꢀreceptor.
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3.1. Synthesis of sigma-2 receptor ligands. Two structurally different synthetic molecules
are proposed as sigmaꢀ2 receptor ligands, namely TA6 and MLP66, that are 6ꢀaminoꢀ
hexyloxy derivatives of previously reported PB28 and compound 1, respectively (Figure 1A).
The former is a thoroughly characterized sigmaꢀ2 receptor ligand used as a tool for sigmaꢀ2
receptor studies with its subnanomolar affinity, but no selectivity towards the sigmaꢀ1
receptor. The latter belongs to the recently developed class of sigmaꢀ2 receptor binding
dihydroꢀ(2H)ꢀisoquinolinꢀ1ꢀone derivatives, and it is endowed with a nanomolar affinity and
excellent selectivity for the sigmaꢀ2 subtype (Figure 1B). Interestingly, previous studies
highlighted that the fluorescent compounds F412 and NO1, based respectively on the PB28
and compound 1 scaffold suitably conjugated with green emitting fluorophores, result
excellent tools for sigmaꢀ2 receptor studies, as they could be visualized by means of flow
49ꢀ50
cytometry and confocal microscopy in live cells (Figure 1B).29,
The improved
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pharmacological (Figure 1B) and fluorescent properties of the two fluorescent compounds
F412 and NO1, with respect to the previously reported sigmaꢀ2 fluorescent ligands,49 were
successfully achieved in the structure of PB28 and compound 1 by replacing the methoxy
group by a hexamethylenoxy chain, and thus identifying the 6ꢀmethylenes chain as an optimal
spacer between the sigmaꢀ2 pharmacophore and the fluorescent moiety. According to these
findings, the 6ꢀaminoꢀhexamethylenoxy arm was here attached at 5ꢀposition on the tetralin
ring for PB28 and on 6ꢀposition of the isoquinolinꢀ1ꢀone for compound 1 analogue, providing
also the amine group as functional group for the subsequent covalent binding of the ligand
with the primary amine groups at the NP silica surface. As in F412 and NO1, derivatization of
PB28 and compound 1 with a long spacer from the silicaꢀcoated QDs is expected to preserve
their affinity and selectivity to sigmaꢀ2 receptors.49ꢀ50
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Figure 1. (A) Structures of sigmaꢀ2 lead compounds (1 and PB28) and fluorescent sigmaꢀ2
ligands (NO1 and F412); (B) Table of the inhibition constants (Ki) at both sigma receptor
subtypes (sigmaꢀ1 and sigmaꢀ2) for lead compounds and fluorescent ligands. a From ref. 45;
b From ref. 44; c From ref. 50; d From ref. 49.
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In particular, the synthesis of MLP66 is reported in Scheme 1, while the synthesis of TA6
was achieved through a previously reported pathway.44 The box in the Scheme 1 reports the
synthesis of the key mesylate intermediate 3, achieved by initial protection of the amine
function of the 6ꢀaminoꢀ1ꢀhexanol by condensation with acetonylacetone to a 2,5ꢀ
dimethylpyrrole ring (2), followed by mesylation of the primary alcohol with mesyl chloride.
The 6ꢀhydroxyꢀ3,4ꢀdihydroꢀisoquinolinꢀ(2H)ꢀ1ꢀone (4) was obtained according to a
procedure45 previously reported for a different isomer. Benzylation of the phenolic function
led to the benzyl derivative,51 which underwent alkylation with 1ꢀbromoꢀ3ꢀchloroꢀpropane in
the presence of NaH to generate intermediate 6. Reaction of this last compound with 6,7ꢀ
dimethoxytetrahydroisoquinoline led to compound 7, whose debenzylation by H2 in the
presence of Pd on activated charcoal 10% led to the phenol intermediate 8. Alkylation of 8
with intermediate 3 provided compound 9 that upon deprotection with NH2OH provided the
amine derivative MLP66.
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Scheme 1. Reaction scheme of the synthesis of MLP66 ligand and of the mesylate
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intermediate (3, left box).
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While the UVꢀVis absorption spectra for both the sigmaꢀ2ꢀligands are quite similar,
characterized by an intense absorption signal below 350 nm ascribed to the aromatic πꢀ π*
transition, the spectrofluorometric investigation (λex= 300 nm) points out the specific PL
signature of TA6 (Figure 2A) and MLP66 (Figure 2B), respectively. In particular, the
fluorescence spectrum of TA6 shows an emission band centered at 345 nm ascribed to the
tetralin functionality (Figure 2A), while the tetrahydroisoquinoline moiety of MLP66 (Figure
2B) generates a remarkable intense band at 465 nm with a shoulder at 375 nm.
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Figure 2. UVꢀVis absorbance and emission spectra, λex = 300 nm, of TA6 (A) and MLP66
(B), along with the molecular structures.
3.2 Synthesis and characterization of silica-coated QDs functionalized with sigma-2
receptor ligands
Hydrophobic, red emitting QDs (See Figure S1, Supporting Information for spectroscopic
and morphologic characterization) were coated with a silica shell by using a microemulsion
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based strategy, resulting in hydrophilic, coreꢀshell type QD@SiO2 NPs, with a surface that
could be promptly functionalized.13,43,47
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Figure 3. UVꢀVis absorbance and PL emission (λex = 400 nm) spectra (A) and transmission
electron micrograph (B) of silicaꢀcoated QDs.
According to this procedure, the spectroscopic properties of the QDs48 were effectively
conveyed to the resulting silica coated nanostructures, that retained the characteristic wide
absorption spectrum of QDs and the narrow emission band centred at 613 nm (Figure 3A). In
addition, TEM analysis revealed the formation of homogeneous and monodisperse coreꢀshell
structures presenting multiple QD cores embedded in a silica shell, with a spherical
morphology of nearly 32 nm diameter (σ%= 15%, Figure 3B).
Prior to functionalization with the sigmaꢀ2 receptors ligands, primary amine (NH2) groups
were grafted onto the nanoparticle silica surface by reaction with 3ꢀ
aminopropyltriethoxysilane, thus providing effective anchor sites for their subsequent
covalent binding to the primary amine groups present in the two distinct ligand structures
(Chart 1). TA6 and MLP66 were covalently anchored to the silica surface, in the presence of
BS3, as shown in Figure 4, where the characteristic emission bands for the two ligands
(Figure 4 A1, B1, λex = 300 nm) are clearly visible together with the PL fingerprint of the QDs
(Figure 4 A2, B2, λex = 400 nm), thus clearly indicating the successful conjugation of the
ligands onto the luminescent NP silica surface. The emission intensity characteristic of the
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two sigmaꢀ2 receptors ligands (Figure 4 A1 and B1) conjugated to luminescent silicaꢀbased
NPs, allowed to estimate the concentration of each conjugated ligand in the sample, as
described in the experimental section. Starting from 0.5 mg/mL of QD@SiO2ꢀNH2 NPs
suspension containing 2 µmol of NH2 groups, 8.0 µg/mL (± 0.5 µg/mL) and 10.0 µg/mL (±
0.5 µg/mL) for TA6 and MLP66 ligand, respectively, were estimated.
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A mixture containing amine functionalized silicaꢀcoated QDs and the free sigmaꢀ2 receptor
ligands was incubated, under the same experimental conditions for conjugation reaction but
without any linker, in order to demonstrate that no PL contribution arises from nonꢀspecific
absorption of the two ligands at the surface of QD@SiO2ꢀNH2 NPs. Indeed, any PL feature
characteristic of the two ligands was observed in the emission spectrum of the purified
mixture, thus indicating that the PL signals of the two ligands in the corresponding sample
spectrum can be detected only thank to their covalent conjugation with amine groups at NP
surface which occurs in the presence of crosslinking agents (see Supporting Information,
Figure S2).
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Figure 4. Sketches of the QD@SiO2ꢀNH2 NPs functionalized with TA6 (Panel A) and
MLP66 (Panel B) along with the corresponding fluorescence spectra (λex= 300 nm, A1, B1;
λex= 400 nm, A2, B2) and TEM micrographs (A3, B3).
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The retention of the peculiar optical properties of luminescent QDs can be unequivocally
confirmed. The slight decrease observed in the absolute PL QY of the colloidal nanostructures
dispersed in aqueous solution and measured at 400 nm (nearly 8% and 7%, for the
nanostructure conjugated with MLP66 and TA6, respectively) with respect to the QY of the
as-synthesized QDs (nearly 11%) can be reasonably ascribed to the change in the surface
chemistry upon functionalization reaction and dispersant media (DMSO or water compared to
the pristine hexane). Interestingly, these QY values for the MLP66ꢀ and TA6ꢀconjugated
luminescent silicaꢀbased nanostructures are significantly higher than those recorded for the
overall so far reported organic sigmaꢀ2ꢀ targeting fluorescent ligands (that show an extremely
low QY value in aqueous solutions,49,50) highlighting the promising impact of the prepared
hybrids nanostructures as efficient nanoprobes for the optically traceable sigmaꢀ2 receptor
targeting.
In addition, the morphological investigation of the two luminescent nanostructured conjugates
was carried out by means of Transmission Electron Microscopy (TEM), Dynamic Light
Scattering (DLS) analysis and ζꢀpotential measurements, since cellular uptake, membrane
adsorption, membrane transport, as well as the fate of the NPs in the cell strongly depend on
NP size, morphology and surface charge.
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Figure 5. Size distribution obtained by DLS (A) and ζꢀpotential measurements (B) of
QD@SiO2 NPs (black line), amino functionalized QD@SiO2 NPs (red line), MLP66ꢀ
conjugated QD@SiO2 NPs (green line) and TA6ꢀconjugated QD@SiO2 NPs (blue line), along
with a sketch of the silica surface.
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No significant changes in the silicaꢀbased NP morphology and in their average size were
detected by TEM analysis after conjugation with the two different ligands (Figure 3 A3, B3),
resulting in NPs of nearly 33 nm (σ% = 16%, Figure 4A3) and 32 nm (σ% = 15%, Figure 4
B3).
DLS analysis and ζꢀpotential measurements were performed to record hydrodynamic diameter
and surface charge density of the luminescent QD@SiO2 NPs, at each functionalization steps,
starting from the “as synthesized” QD@SiO2 NPs, to amineꢀfunctionalized QD@SiO2 NPs,
and finally to MLP66ꢀ or TA6ꢀconjugated QD@SiO2 NPs (Figure 5). DLS analysis clearly
indicated, for each step, a monomodal size distribution, thus suggesting that no aggregation
phenomenon of silicaꢀbased nanostructure occurs in aqueous medium. In particular, average
diameters of 36 (PDI 0.261 ± 0.016), 37 (PDI 0.169 ± 0.019), 41 (PDI 0.341 ± 0.025) and 43
nm (PDI 0.225 ± 0.011) were recorded for “as synthesized” QD@SiO2 NPs, amineꢀ
functionalized QD@SiO2 NPs, TA6ꢀconjugated QD@SiO2 NPs and MLP66ꢀconjugated
QD@SiO2 NPs, respectively. While no significant change in hydrodynamic diameter was
observed passing from QD@SiO2 NPs to amine functionalized QD@SiO2 NPs, a slightly
increase was recorded after silica NP conjugation with MLP66 or TA6 (Figure 5A). The size
measured for sigmaꢀ2 receptor ligand conjugatedꢀQD@SiO2 NPs with the TEM analysis (33
and 32 nm for TA6 and MLP66 conjugated nanostructures, respectively, Figure 4) differed
from the DLS measurements, being TEM observation, in the here tested experimental
conditions, able to detect only the inorganic domain of the hybrid conjugated nanostructure.
Conversely, the higher hydrodynamic diameter values resulting from the DLS investigation,
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(namely 41 and 43 nm for TA6ꢀconjugated QD@SiO2 NPs and MLP66ꢀconjugated
QD@SiO2 NPs, respectively, Figure 5A blue and green line), resulted, beside to the inorganic
component, from the presence of the MLP66ꢀ or TA6ꢀlinked to NP surface, the hydration
shell, and also the excess of counter ions.
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ζꢀpotential measurements provided insights on the colloidal stability, as well as on the surface
charge of the luminescent NPs at each step of the functionalization process. Upon grafting the
amine groups, the negatively charged QD@SiO2 NPs (ꢀ45.0 ± 2.0 mV, Figure 5B black line)
become positive (28.6 ± 0.3 mV, Figure 5B red line), in agreement with the literature reported
values.17 Further conjugation with each of the two sigmaꢀ2 receptor ligands results in an
average ζꢀpotential value of ꢀ20.0 ± 0.7 mV and ꢀ19.4 ± 0.8 mV for TA6 (Figure 5B, blue
line) and MLP66 (Figure 5B, green line), respectively. The less positive values of QD@SiO2
NPs functionalized with the sigmaꢀ2 receptors ligands, with respect to that of the amino
functionalized sample, can be ascribed to the decrease of the amount of the free amino groups
present at the surface. In details, the functionalization with either TA6 or MLP66 induces a
partial charge neutralization, thanks to the formation of a covalent bond between the surface
amine groups and the targeting moiety, mediated by the BS3 spacer. Such an evidence further
confirmed the success of the conjugation reaction.
3.3 Cell viability
The two types of QD@SiO2 NPs conjugated with TA6 and MLP66 (QD@SiO2ꢀTA6 and
QD@SiO2ꢀMLP66 NPs) and the unconjugated QD@SiO2ꢀNH2 NPs were studied in human
breast adenocarcinoma cells (MCF7). This cell type was selected, as previously and
thoroughly characterized, for the presence of sigmaꢀ2 receptor, thus resulting in a wellꢀ
established model for sigmaꢀ2 receptor studies.50, 52 As a first step, the cytotoxic activity of
QD@SiO2 NPs was evaluated after longꢀterm exposure (72 hours).
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The experimental data indicated that the QD@SiO2ꢀMLP66 (EC50 = 0.007 mg/mL ±0.001,
Figure 6A green line) and QD@SiO2ꢀTA6 (EC50 = 0.008 mg/mL ±0.001, Figure 6A blue
line) NPs resulted in a slightly higher toxicity in MCF7 cells than the bare, unconjugated,
QD@SiO2ꢀNH2 NPs (EC50 = 0.015 mg/mL ±0.001 Figure 6A red line), due to the presence
of the sigmaꢀ2 receptorꢀtargeting moiety on the conjugated NP surface, as expected. Indeed,
although mechanisms underlying cell death are not fully disclosed, inhibition of the sigmaꢀ2
receptor has been reported to result in caspaseꢀdependent and/or caspaseꢀindependent
apoptosis, lysosome leakage, cathepsin activation, autophagy and oxidative stress.53
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Figure 6. (A) Concentration–cell viability profiles for MCF7 cancerous cells after 72 h of
incubation with QD@SiO2ꢀNH2 (red line and symbol), QD@SiO2ꢀTA6 (blue line and
symbol) and QD@SiO2ꢀMLP66 (green line and symbol) NPs. Each point represents a mean
value of three independent experiments. (B) MCF7 cell uptake by flow cytometry analysis
versus time after 3 h, 5 h or 24 h.
According to the results obtained from cytotoxicity assay, the concentration of 0.010 mg/ml
was selected and used for all tested NPs (QD@SiO2ꢀNH2, QD@SiO2ꢀMLP66 and QD@SiO2ꢀ
TA6 NPs), to perform flow cytometry, confocal, as well as live cell microscopy experiments.
At this concentration, the sigmaꢀ2 targeted nanosystems have a concentration of nearly 500
nM of sigmaꢀ2 receptor affinity ligands.
3.4 Flow cytometry investigation
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The fluorescence signals originated from internalized luminescent NPs allowed to
quantitatively evaluate their cell uptake at three different incubation times. In particular,
MCF7 cell lines were treated with QD@SiO2ꢀNH2, QD@SiO2ꢀMLP66 or QD@SiO2ꢀTA6
NPs, at a concentration of 0.010 mg/mL, for 3 h, 5 h or 24 h (Figure 6B). The obtained
results clearly indicated cellular uptake of targeted and nonꢀtargeted luminescent silicaꢀbased
NPs, thus highlighting the timeꢀdependence of the extracellular membrane crossing and
penetration.
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While QD@SiO2ꢀNH2 NPs (Figure 6B, red bars) could already be detected inside the cells
after 3 h, the conjugated QD@SiO2ꢀTA6 (Figure 6B, blue bars) and QD@SiO2ꢀMLP66
(Figure 6B, green bars) NPs took longer to enter the cells. This behaviour can be explained by
assuming different pathways involved for the cell internalization for the targeted and nonꢀ
targeted NPs.
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Figure 7. Representative single central confocal slice of fixed MCF7 cells imaged incubated
with QD@SiO2ꢀNH2 (AꢀC), QD@SiO2ꢀTA6 (A1ꢀC1) and QD@SiO2ꢀMLP66 (A2ꢀC2) NPs
for 24 h and stained with DAPI. Red (A, A1, A2), blue (B, B1, B2) and overlays detection
channel (C, C1, C2). Scale bar 20 nm.
The surface charge shift from positive to negative values passing from QD@SiO2ꢀNH2 to
QD@SiO2ꢀMLP66 or QD@SiO2ꢀTA6 NPs, respectively, may contribute to explain their
different behaviour in the cell internalization process. NPs smaller than 50 nm in diameter and
>10 mV surface charge were reported to undergo an enhanced cellular internalization through
a nonspecific process, namely adsorptive endocytosis,54 and that cellular uptake of anionic
NPs can be slower than that of cationic NPs.55
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Indeed, as electrostatic interactions are considered important in NP−cell interaction, it is
reasonable to assume that the internalization of positive charges at the surface of QD@SiO2ꢀ
NH2 (with ≤ 50 nm size and >10 mV surface charge) can be promoted by binding to the large
negatively charged domains which are present at the cell surface thus initiating their rapid
uptake, mainly via predominant clathrinꢀmediated endocytosis.54,56 Conversely, the observed
cellular uptake of negatively charged and targeted QD@SiO2ꢀMLP66 or QD@SiO2ꢀTA6 NPs
could involve their adsorption to the few cationic sites on the cell surface. Importantly, a
receptorꢀmediated endocytosis cannot be completely excluded, since previous studies proved
that sigmaꢀ2 receptors exist in lipid rafts, which are organized microdomains in cell
membranes, largely found in the plasma membrane.53
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Therefore, according to the experimental findings obtained by flow cytometry measurements,
the confocal microscopy experiments were subsequently performed after 24 h incubation
(Figure 7).
3.5 Confocal Microscopy Investigation
Here MCF7 cells, after 24 h incubation with the 0.010 mg/mL of redꢀemitting QD@SiO2ꢀNH2
(Figure 7AꢀC), QD@SiO2ꢀTA6 (Figure 7A1ꢀC1) and QD@SiO2ꢀMLP66 (Figure 7A2ꢀC2) NP
samples, were fixed and stained with DAPI, (blueꢀemitting dye, generally used to stain nuclei
of fixed cells, since it is cellꢀimpermeant) to attain preliminary information on the
intracellular distribution of the nanostructures. Images at different focal planes were recorded
in order to render and visualize the entire sample volume. In particular, ten “Z stacks” were
registered by incrementally stepping through the fixed cell samples using a focal drive, and in
Figure 7 the central stack acquired for the fixed MCF7 cells incubated with the QD@SiO2ꢀ
NPs (red) and stained with DAPI (blue) are reported. The analysis revealed that redꢀemitting
spots are clearly visible in the red detection channel and are ascribed to the QD@SiO2
nanostructures (Figure 7 A, A1, A2). The merge pictures (Figure 7 C, C1, C2), that reports the
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overlapping of the red detection channel with the blue ones used to visualize the DAPI
(Figure 7 B, B1, B2), show the red emitting spots close to the blueꢀemitting features (nuclei).
The image confirms the amineꢀ and sigmaꢀ2 ligandsꢀfunctionalized nanostructures uptake, in
agreement with the flow cytometry experiments, although coꢀlocalization of the two targeted
luminescent nanostructures in the nucleus was not observed, proving that none of the three
tested silica nanoparticles were localized in the nuclei. In order to investigate, whether the
targeted NPs coꢀlocalize with sigmaꢀ2 receptor, coꢀlocalization experiments by coꢀincubated
0.010 mg/mL of functionalized QD@SiO2 NPs and 10 µM of greenꢀemitting sigmaꢀ2 receptor
agonist NO1 (Figure 8, experiments were also performed with F412 at 10 µM, data not
shown) were conducted using live MCF7 cells. Confocal microscopy imaging in live cells
represent an imaging modality able to give a more accurate picture of the intracellular
location of QD@SiO2 NPs.
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While the more diffusible small fluorescent sigmaꢀ2 ligand NO-1 thoroughly distributes in the
cytoplasm in accordance with the sigmaꢀ2 receptor localization at diverse cytoplasmic
organelles, the rather large QDs appear to be less distributed.53 In addition, the overlay
channels reported in Figure 8 CꢀC2 reveal that while for QD@SiO2ꢀNH2 no coꢀlocalization
with NO1 occur (Figure 8C, see also Figure S3a in the Supporting Information), a not
negligible number of yellow spots ascribed to coꢀlocalization of redꢀemitting QD@SiO2ꢀTA6
or QD@SiO2ꢀMLP66 NPs with greenꢀemitting NO1 are clearly visible in Figure 8C1ꢀC2 (see
also Figure S3bꢀc in the Supporting Information). This highlights a similar intracellular fate
after the cell internalization of both the small fluorescent sigmaꢀ2 ligand and targeted
luminescent NPs, finally supporting their comparable targeting affinity for sigmaꢀ2 receptor.
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Figure 8. Confocal microscopy analysis showing live MCF7 cells stained with the
fluorescent sigmaꢀ2 receptor ligand NO1 (10 µM, green), and QD@SiO2ꢀNH2 (AꢀD),
QD@SiO2ꢀTA6 (A1ꢀD1) or QD@SiO2ꢀMLP66 (A2ꢀD2) NPs (0.010 mg/ml, red). Green (A,
A1, A2), red (B, B1, B2) along with the overlay (C, C1, C2) and bright field detection
channels (D, D1, D2). Scale bar=20 ꢁm. Each image is representative of one experiment out
of three.
The absence of more important portions of coꢀlocalization may be justified by the different
kinetics of the small fluorescent ligand saturating the sigmaꢀ2 receptors. In addition, it is
worth to note that while NO1 sigmaꢀ2 ligand used for incubation with MCF7 cells in live cells
experiments had a concentration of 10 µM, in the case the targeted NPs (QD@SiO2ꢀTA6 and
QD@SiO2ꢀMLP66) a concentration of 0.010 mg/mL was exploited, which corresponds to a
ligand concentration at NP surface of nearly 500 nM (as estimated from the spectroscopic
characterization). This means that the NO1 agonist, used for cells experiments, was about 20
times more concentrated than the ligands, conjugated to NP silica surface. While the
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