Clicking porphyrins to magnetic nanoparticles for photodynamic therapy

Article abstract of DOI:10.1002/cplu.201300276

A method for the preparation of superparamagnetic iron oxide nanoparticle-porphyrin (SPION-TPP) conjugates through click chemistry, which can be used as novel theranostic nanoagents for photodynamic therapy is developed. The synthesis, characterisation, and evaluation of the photocytotoxicity profiles of the nanoconjugates prepared is reported. Upon light irradiation, SPION-TPP nanoconstructs promote a photodynamic effect in vitro in murine amelanotic melanoma B78-H1 cells, with IC50 values in the region of 800 nm, similarly to unbound TPP, whereas they remain non-cytotoxic in the dark. However, these nanoconstructs show poor cellular uptake, which influences a linear dose-response effect. Therefore, the improvement of delivery to cells has also been studied by conjugating a well-known cell-penetrating peptide (TAT peptide) to the SPION-TPP nanoparticles. The new nanoconstructs show lower IC50 values (in the region of 500 nm) and a clear dose-response effect. Our results suggest that TAT-conjugated SPION-TPP nanoparticles are efficient nanodevices both for tracking drugs by means of magnetic resonance imaging (MRI)-based techniques and for treating cancer cells through photodynamic therapy, thus functioning as promising theranostic nanoagents.

Full text of DOI:10.1002/cplu.201300276

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DOI: 10.1002/cplu.201300276
“
Clicking” Porphyrins to Magnetic Nanoparticles for
Photodynamic Therapy
[a]
[c]
[c]
[b, d, e]
Merlyn Thandu, Valentina Rapozzi, Luigi Xodo, Fernando Albericio,*
Clara Comuzzi,* and Silvia Cavalli*
[a]
[a, b, f]
A method for the preparation of superparamagnetic iron oxide
nanoparticle–porphyrin (SPION-TPP) conjugates through click
chemistry, which can be used as novel theranostic nanoagents
for photodynamic therapy is developed. The synthesis, charac-
terisation, and evaluation of the photocytotoxicity profiles of
the nanoconjugates prepared is reported. Upon light irradia-
tion, SPION-TPP nanoconstructs promote a photodynamic
effect in vitro in murine amelanotic melanoma B78-H1 cells,
with IC50 values in the region of 800 nm, similarly to unbound
TPP, whereas they remain non-cytotoxic in the dark. However,
these nanoconstructs show poor cellular uptake, which influen-
ces a linear dose–response effect. Therefore, the improvement
of delivery to cells has also been studied by conjugating
a well-known cell-penetrating peptide (TAT peptide) to the
SPION-TPP nanoparticles. The new nanoconstructs show lower
IC50 values (in the region of 500 nm) and a clear dose–response
effect. Our results suggest that TAT-conjugated SPION-TPP
nanoparticles are efficient nanodevices both for tracking drugs
by means of magnetic resonance imaging (MRI)-based tech-
niques and for treating cancer cells through photodynamic
therapy, thus functioning as promising theranostic nanoagents.
Introduction
Advances in nanotechnology have led to new opportunities
for the detection, cure, and post-treatment monitoring of
promoting the simultaneous treatment and diagnosis of dis-
eases, as well as determining the distribution, release, and
[
1]
[2]
cancer. In this regard, theranostic nanoagents are advanced
tools in nanomedicine; they combine drugs and probes within
the same nanostructure, and thus, have unique potential in
therapeutic efficacy of a drug. Theranostics based on engi-
neered superparamagnetic iron oxide nanoparticles (SPIONs)
are particularly interesting. Because of their superparamagnetic
character, SPIONs can be used as negative contrast agents in
[
a] M. Thandu, Dr. C. Comuzzi, Dr. S. Cavalli
Department of Chemistry, Physics and Environment
University of Udine
[2,3]
magnetic resonance imaging (MRI). Furthermore, upon func-
tionalisation of SPIONs with fluorescent dyes, they can act as
bimodal nanodevices for both magnetic resonance and fluo-
Via del Cotonificio 108, 33100 Udine (Italy)
E-mail: clara.comuzzi@uniud.it
[4]
rescence microscopy imaging. Moreover, they can be used
[2a]
[b] Prof. Dr. F. Albericio, Dr. S. Cavalli
for hyperthermia therapy (HT),
a technique in which the
CIBER-BBN
magnetic particles are heated selectively by applying a high-
frequency magnetic field and then used to achieve thermal
ablation of tumours, or as magnetic vectors that can be direct-
ed to a specific target by exploiting a magnetic field gradi-
Networking Centre on Bioengineering, Biomaterials, and Nanomedicine
Institute for Research in Biomedicine (IRB-Barcelona)
Baldiri Reixac 10, 08028 Barcelona (Spain)
E-mail: albericio@irbbarcelona.org
[5]
[c] Dr. V. Rapozzi, Prof. Dr. L. Xodo
ent.
Department of Medical and Biological Sciences
School of Medicine, University of Udine
Piazzale Kolbe 4, 33100 Udine (Italy)
Photodynamic therapy (PDT) is a local clinical treatment of
cancers, and is particularly efficient in the case of skin tumours.
PDT involves three nontoxic components: a photosensitiser,
[
d] Prof. Dr. F. Albericio
[6]
Department of Organic Chemistry
University of Barcelona, 08028 Barcelona (Spain)
light, and oxygen. In combination, these elements give rise
to cytotoxic oxygen species such as singlet oxygen, which
result in cell death. In this medical practice, photosensitising
molecules play a crucial role in generating singlet oxygen after
light irradiation to cause oxidative damage, which leads to the
[
e] Prof. Dr. F. Albericio
School of Chemistry and Physics
University of KwaZulu-Natal, 4001 Durban (South Africa)
[
f] Dr. S. Cavalli
[7]
death of malignant cells. Porphyrins and their derivatives
have been studied extensively as powerful photosensitisers.
Their unique photophysical and photochemical properties and
presence in natural systems make them an attractive choice
Current address:
Center for Advanced Biomaterials for Health Care IIT@CRIB
Istituto Italiano di Tecnologia
Largo Barsanti e Matteucci 53
8
0125 Naples (Italy)
[8]
for the generation of singlet oxygen in PDT. Some commer-
cially available porphyrins (e.g., Photofrin) are already used for
E-mail: silvia.cavalli@iit.it
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201300276.
[7a,9]
tumour treatment.
There is great research interest in the
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possibility of synthesising novel porphyrin derivatives for use
quence, Rhod-TAT-SPION-TPP nanoagents could also be used
as probes for confocal microscopy, and allow for the subcellu-
lar localisation of the photosensitiser magnetic nanoparticles.
The efficiency of singlet oxygen production upon light irradia-
tion was studied through UV/Vis spectroscopy and compared
to that of the unbound TPP derivative. Subsequently, detailed
biological studies were conducted to evaluate the cytotoxicity
and cellular uptake of the new synthesised nanoconstructs to
validate them as useful theranostics for photodynamic therapy.
[10]
in targeted tumour therapy and diagnosis (theranostics). An-
other important feature of novel photosensitisers is their ca-
pacity to absorb at wavelengths in the red region, which is
[11]
most penetrating in living tissue (therapeutic window). Fur-
thermore, numerous methodologies for active targeting have
been developed to increase the efficacy of photosensitisers,
[12]
such as their conjugation to peptides or antibodies.
PDT
agents have also been encapsulated within polymers to yield
[
13]
higher local concentrations at target sites.
Nanoparticles
have also been used to enhance the delivery of hydrophobic
[
14]
photosensitisers. For example, the conjugation of porphyrins Results and Discussion
to magnetic nanoparticles has been shown to increase their
Synthesis and characterisation
cellular uptake. Moreover, porphyrins are combined with bio-
logical vectors such as proteins, steroids, toxins, carbohydrates,
peptides and functionalised nanoparticles to overcome the
problem of systemic prolonged photosensitisation syndrome
A short ethylene glycol derivative of TPP (compound 4 in
Scheme 1) was synthesised to increase the water affinity of the
photosensitiser and favour its conjugation to SPION through
“click chemistry” in buffer media.
[
15]
shown by healthy tissues.
The so-called “click” reaction, or copper(I)-catalysed Huisgen
,3-dipolar cycloaddition, is of considerable relevance as a con-
In the first step of the synthesis, propargyl amine was react-
ed to commercially available 2-[2-(Boc-amino)ethoxy]ethoxy-
acetic acid (dicyclohexylammonium salt) with N’-(3-dimethyla-
minopropyl)-N-ethylcarbodiimide (EDC), 1-hydroxybenzotria-
zole (HOBt) and triethylamine (TEA) in anhydrous dichlorome-
thane (DCM). The condensed product was then treated with
a 1:1 (v/v) solution of trifluoroacetic acid (TFA) in DCM to
remove the Boc protecting group. Subsequently, the product
was reacted directly with TPP using similar reaction conditions
to those in the first step of the synthesis.
1
jugation method for a wide range of biomolecular applications
because of the unreactive nature of both azides and alkynes
[16]
towards functional groups that are present in biomolecules.
Moreover, the thermal and hydrolytic stability of the cycloaddi-
tion product is of great interest in the field of bioconjugation.
In addition to the chemoselectivity and stability of the “click”
reaction, it is also particularly interesting that it occurs effi-
ciently in aqueous media at room temperature and with high
selectivity, as only the 1,5-substituted triazole is formed. This
technique has been used extensively for a broad range of bio-
conjugations such as the preparation of drug-delivery nanosys-
Commercially available SPION-NH nanoparticles were func-
2
tionalised with the NHS ester of 12-azido-4,7,10-trioxadodeca-
noic acid in citrate buffer (pH 8) containing 10 mL of diisopro-
[
17]
[18]
[19]
[21b]
tems, biomaterials, and radiopharmaceutical drugs. The
click” reaction has also been used in the specific field of por-
phyrin synthesis and for the binding of these compounds to
pylethylamine (DIPEA), as shown in Scheme 2.
“Click”
“
chemistry was employed to conjugate the TPP derivative 4 to
azido-functionalised SPION (SPION-N ). Samples were prepared
3
[
20]
materials. Moreover, azido-modified nanoparticles have been
in phosphate buffer at pH 7.5 by using 0.5 equivalents of
CuSO ·5H O and 2 equivalents of Na ascorbate to reduce
[
21]
prepared for general labelling through “click” chemistry.
4
2
Here, we report the synthesis and characterisation of nano-
conjugates between SPIONs and a porphyrin (a derivative of
tetraphenylporphyrin, abbreviated as TPP in the text). Conjuga-
tion was achieved by exploiting copper(I)-mediated “click”
chemistry. In this way, a new nanoconstruct (here abbreviated
as SPION-TPP), which can act as an imaging probe (e.g., con-
trast agents for MRI) as well as a therapeutic drug for anticanc-
er PDT, was prepared. We anticipate that the conjugation of
a cell-penetrating peptide (CPP) to nanoparticles bearing the
photosensitisers may lower the IC values by improving their
in situ copper(II) to copper(I), the effective catalyst in the reac-
tion. After overnight rotation to ensure good mixing of the col-
loidal dispersion, the samples were dialysed first against water
containing 10 mm EDTA to remove copper salt traces, and sub-
sequently against deionised water. A final dialysis against PBS
was performed in the case of samples used for the biological
experiments. Although copper cations were shown to be easily
[20c]
complexed by porphyrins,
we did not observe interference
with the “click” reaction under our experimental conditions.
50
[
22]
cellular uptake. Therefore, a CPP was also conjugated to the
SPION-TPPs to improve delivery to cells. For this reason,
a more advanced nanoconstruct (here abbreviated as Rhod-
TAT-SPION-TPP) was prepared by first functionalising SPIONs
with a well-known CPP (a derived sequence of TAT peptide
functionalised with rhodamine). For this conjugation, we used
a protocol based on the aniline-catalysed oximation described
Analysis
UV/Vis spectroscopy of the nanoconjugate solutions after dial-
ysis showed the occurrence of the Soret band characteristic of
the porphyrin (418 nm), whereas no UV absorbance was ob-
served in the control samples in which no catalyst was added
(data not shown). Furthermore, the occurrence of the reaction
could be checked easily, as upon conjugation, the four Q
bands of the free TPP (at 515, 548, 585, and 641 nm) collapsed
into only one Q band centred at 538 nm (see Supporting Infor-
mation, Figure S1A and D), whereas the Soret band (422 nm)
[
23]
previously by our group for similar purposes. Subsequently,
the Rhod-TAT-SPIONs were linked to TPP through “click chemis-
try”, as in the case of the SPION-TPP nanoparticles. Moreover,
through exploitation of the rhodamine inserted in the TAT se-
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of TPP to the SPION did not alter
1
the O production of unbound
2
TPP (see Figure 2).
With the aim of improving cel-
lular uptake and studying the
cellular internalisation of the
SPION-TPP conjugates, we also
prepared a more advanced type
of nanostructure, Rhod-TAT-
SPION-TPP,
by
conjugating
SPION-TPP to a cell-penetrating
peptide (CPP). As the CPP, we
used the HIV TAT-derived se-
quence: a small basic peptide
(
RKKRRQRRR) that has been
shown to deliver a variety of car-
[26]
goes into cells.
SPIONs were
loaded with both benzaldehyde
and azide (Ald-SPION-N ) react-
3
ing groups, as shown in
Scheme 2. Ald-SPION-N3 nano-
conjugates were first reacted
with an amino-oxyacetyl-derived
peptide sequence of TAT (func-
2
Scheme 1. Synthesis of TPP derivative 4. A) 1.2 equiv EDC, 1.2 equiv HOBT·H O, and 2 equiv TEA in dry DCM,
stirred 5 min; 1.2 equiv propargyl amine added and stirred at RT overnight. B) 1:1 (v/v) of TFA in DCM. C) 10 mg of
tionalised
with
rhodamine,
Rhod-TAT), following a protocol
TPP, 1.5 equiv EDC, 1.5 equiv HOBT·H
and stirred at RT overnight.
2
O and 2 equiv TEA in dry DCM, stirred 5 min; 1.5 equiv compound 3 added
[23]
that we described previously.
After dialysis of the Rhod-TAT-
SPION conjugate against water,
did not shift significantly compared to that of free TPP. The
porphyrin loading on the SPIONs was obtained spectrophoto-
metrically through two methods, as described in the Experi-
mental section and Supporting Information (Figure S1C and
D).
the TPP derivative 4 was also introduced through “click”
chemistry, as described in the text.
The photoactivity of the nanoconjugates was evaluated in
murine amelanotic melanoma B78-H1 cells. The cells were in-
cubated with SPION-TPP or Rhod-TAT-SPION-TPP at different
concentrations for 3 h, under conditions of 378C and 5% CO2,
and subsequently illuminated with a white halogen lamp at
The incidence of the reaction and the formation of the tria-
1
zole ring upon conjugation was also verified through H high-
[24]
À2
resolution magic angle spinning (HRMAS) NMR spectroscopy.
a fluence of 14 Jcm . First, resazurin assays were conducted
As shown in the NMR spectrum in Figure 1, the CH of the tria-
zole was identified as the singlet peak at 9.40 ppm. Further-
more, as expected, the peaks of the pyrrole and phenyl groups
related to the porphyrin molecule were observed in the aro-
matic region (from 7.19 to 8.71 ppm) and at negative ppm
at two concentrations (400 and 800 nm) of SPION-TPP (Fig-
ure 3A). Upon light irradiation, the SPION-TPP nanoconstructs
reduced the metabolic activity of the B78-H1 cells up to
almost 50% compared with the control (nanoparticles without
porphyrin, SPION-NH ). From this experiment, an IC value of
2
50
(
À2.98 ppm). Finally, the peaks related to the CH O group of
about 800 nm can be estimated. As expected, B78-H1 cells
treated with SPION-TPP, but not irradiated with light, did not
show any reduction in metabolic activity, demonstrating that
the nanoparticles were not bioactive or cytotoxic in the dark
(at least at the concentrations used). As anticipated from the
singlet-oxygen generation test, the bioactivity of SPION-TPP
nanoparticles was comparable to that of TPP alone (see Fig-
ure S3 in Supporting Information). However, we did not ob-
serve a clear linear dose–response effect with SPION-TPP (data
not shown). This observation may be attributed to poor cellu-
lar uptake of the nanoparticles and their consequent aggrega-
tion outside the cell membrane. In light of these results, we
decided to increase the uptake of the SPION-TPP nanoparticles
by conjugating them to the HIV TAT-derived sequence. In the
case of this new nanoconstruct (Rhod-TAT-SPION-TPP), we
2
the short ethylene glycol linker were found between 4.39 and
4
.82 ppm. The remaining peaks (0.5–4 ppm) were ascribed to
the dextran-coated SPION and to the deuterated DMSO used
as solvent for the NMR experiments (in addition, the usual
broad peak of water related to the solvent was found at
around 3.4 ppm). No porphyrin-related peaks were found in
1
the control H HRMAS NMR of SPION-N (see Figure S2 in Sup-
3
porting Information).
The capability of SPION-TPP conjugates to generate singlet
oxygen, the key toxic species that provokes the photokilling
effect in PDT, was evaluated through an anthracene-9,10-dipro-
[
14a,25]
pionic acid (ADPA) test,
commercially available
0,15,20-triphenyl-21H,23H-porphyrin; TPP). The conjugation
and was compared to that of the
porphyrin (5-(4-carboxyphenyl)-
1
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Scheme 2. Synthesis of SPION-TPP and Rhod-TAT-SPION-TPP nanoconjugates: A) 5 mg N-hydroxysuccinimidyl ester of 12-azido-4,7,10-trioxadodecanoic acid,
0 mL DIPEA in 0.1m citrate buffer at pH 8, rotated overnight at RT. A’) 5 mg N-hydroxysuccinimidyl ester of 12-azido-4,7,10-trioxadodecanoic acid, 4 mg N-hy-
droxysuccinimidyl ester of 4-formylbenzoic acid, 10 mL DIPEA in 0.1m citrate buffer at pH 8, rotated overnight at RT. B) 2 mg amino-oxyacetyl-derived Rhod-
1
TAT peptide, 100 mm aniline in 0.1m phosphate buffer at pH 7.5, rotated overnight at RT in dark. C) 2 mg TPP derivative 4, 0.5 equiv. CuSO
sodium ascorbate in 0.1m phosphate buffer at pH 7.5, rotated overnight at RT (in dark if rhodamine present).
4 2
·5H O, 10 equiv.
indeed observed a linear dose–
response reduction in metabolic
activity as well as a lower IC50
value (about 500 nm) compared
with that for SPION-TPP (Fig-
ure 3B). As expected, the Rhod-
TAT-SPION-TPP was not cytotoxic
in the dark (Figure 3C).
To gain insight into the photo-
killing mechanism triggered by
the designed compounds, we
studied the photodynamically
treated B78-H1 cells stained with
annexin V and propidium iodide
(
PI), through fluorescence-acti-
vated cell sorting (FACS) analysis.
In early apoptosis, cell mem-
branes are known to lose their
phospholipid asymmetry, and
phosphatidyl serine (PS), which
is normally located in the inner
leaflet, redistributes to the outer
leaflet. Because annexin V shows
a high affinity for PS, annexin V-
1
Figure 1. H HRMAS-NMR of SPION-TPP. 2 mg of lyophilised nanoconjugate powder was dispersed in 60 mL of deu-
terated DMSO (1024 scans at RT).
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FITC can mark cells in early apoptosis. In contrast, necrotic
cells, which are permeable to both annexin V and PI, are
[27]
stained by the two dyes.
Figure 4 shows that the treatment of B78-H1 cells with
400 nm SPION-TPP increased the fraction of early apoptotic
and necrotic cells compared with untreated cells (apoptotic
cells from 2.23% to 17.51%; necrotic cells from 2.09% to
7.9%). However, upon treatment of cells with 400 nm of Rhod-
TAT-SPION-TPP nanoparticles, the sum of the fraction of necrot-
ic and apoptotic cells increased up to 35.42%, whereas with
SPION-TPP, the same fraction increased only to 25.41%. TPP
concentrations below IC50 values were selected to observe all
possible cell populations (healthy, necrotic, and apoptotic
cells). These data demonstrated that TAT conjugation was an
efficient strategy for enhancing the efficacy of the SPION-TPP
nanoparticles and decreasing the metabolic activity, and thus
the proliferation, of the B78-H1 cells. This enhancement is
probably caused by a TAT-mediated increase in nanoparticle
cellular uptake. The efficient uptake of Rhod-TAT-SPION-TPP
could also be visualised in the FACS experiments reported in
Figure 4D. Given that rhodamine dye, which is present on the
TAT peptide conjugated to the nanoparticles, absorbs in the
red region (567 nm), the FACS plots showed that the whole
cell population was shifted to this region (upwards along the
y-axis, in comparison to the bottom left panels), indicating that
all the cells had indeed incorporated Rhod-TAT-SPION-TPP
nanoparticles.
Figure 2. Photo-oxidation of anthracene-9,10-dipropionic acid (ADPA). Ex-
periment conducted by exposing solutions containing ADPA and photosen-
sitisers to light (60 W) for different durations. The absorbance decay, caused
1
À1
by O
2
oxidation of ADPA, is reported as lnA
0
A
0
vs. t (s). A is the ADPA ab-
sorbance at 455 nm at t=0, and A is the ADPA absorbance at 455 nm at
t=1–10, corresponding to 900, 1800, 2700, 3600, 4500, 5400, 7200, 9000,
1
0800, and 14400 s, respectively. The treated solutions were: A) 73 mm
ADPA and 0.5 mm 5-(4-carboxyphenyl)-10,15,20-triphenyl-21H,23H-porphyrin
TPP) in PBS (black squares); and B) 73 mm ADPA and 0.5 mm SPION-TPP in
(
PBS (white triangles). TPP contents were calculated according to the UV/Vis
data by applying the method described in the experimental part.
To corroborate this observa-
tion, we performed confocal mi-
croscopy measurements with
Rhod-TAT-SPION-TPP nanoparti-
cles. Figure 5 shows that the
Rhod-TAT-bearing nanoparticles
were taken up quickly by the
cells; after incubation for 3 h,
the cytoplasm of the cells was
strongly stained red owing to
the rhodamine fluorescence. Fur-
thermore, we found that nano-
particles were still present inside
the cells even 24 h after delivery.
Conclusion
We have described a synthetic
route for the preparation of su-
perparamagnetic iron oxide–por-
phyrin (SPION-TPP) conjugates
through “click” chemistry. We
have also reported the charac-
terisation and evaluation of the
phototoxicity of the designed
nanoconjugates. Our results
Figure 3. Phototoxicity of SPION-TPP and Rhod-TAT-SPION-TPP nanoconjugates. Resazurin viability test with B78-
H1 cells. Cells were incubated for 3 h at 378C and 5% CO using increasing concentrations of TPP conjugated to
showed that upon light irradia-
tion, SPION-TPP nanoconstructs
2
the nanoparticles (TPP contents were calculated according to the UV/Vis data by applying the method described
À2
promoted
a
photodynamic
in the experimental part). One set of cells was illuminated with a white halogen lamp at 14 Jcm , and another
set was kept in the dark. Resazurin was added after 23 h, and the readout was obtained 24 h after illumination.
effect in murine amelanotic mel-
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nalisation of Rhod-TAT-SPION-
TPP was confirmed through con-
focal microscopy by exploiting
the rhodamine dye bound to
the TAT peptide sequence.
Furthermore, we previously
demonstrated through MRI ex-
[23]
periments that TAT-functional-
ised SPION nanoparticles could
lower T2 in labelled HeLa cells,
thus functioning as promising
contrast agents (see Figure S4 in
Supporting Information).
In summary, on the basis of
our results, we propose Rhod-
TAT-SPION-TPP nanoparticles as
promising new theranostic nano-
agents: 1) for the treatment of
cancer cells by photodynamic
therapy (we believe that be-
cause of the enhanced permea-
bility and retention effect exhib-
ited by the tumour tissues
against high molecular particles,
the designed nanoconstructs
may show good tumour specific-
Figure 4. FACS analysis of B78-H1 cells treated photodynamically with SPION-TPP and Rhod-TAT-SPION-TPP and
stained with annexin V-FITC and PI. B78-H1 cells were plated at a density of 5ꢁ10 cells in a 6-well plate. After
5
24 h, the cells were A) untreated, B) treated with 300 nm SPION-TPP solution, C) treated with 400 nm SPION-TPP
[28]
ity after blood delivery);
and
solution, and D) treated with 400 nm Rhod-TAT-SPION-TPP solution. After 3 h, the cells were irradiated with light
À2
(
14 Jcm ), and after 24 h they were stained with the components of the Annexin-V-FLUOS staining kit and ana-
2) for the localisation and track-
ing of therapeutic agents
through MRI-based and/or fluo-
lysed by FACS. FL1-H and FL2-H stand for Annexin V FLUOS and propidium iodide (PI) channels, respectively.
rescence-based
techniques.
microscopic
Experimental Section
Materials and methods: All re-
agents and solvents were pur-
chased from commercial sources
and used as received. Nanomag-d-
spio, here referred to as SPION-NH2
À1
(
NH2 surface, 20 nm; 5 mgmL
À1
solid content; 2.4 mgmL
iron
Figure 5. Uptake by confocal laser microscopy. Images of B78-H1 cells untreated (A) and treated with 400 nm
Rhod-TAT-SPION-TPP after incubation for 3 h (B) and 24 h (C).
concentration), was obtained from
Micromod Partikeltechnologie
GmbH, Germany. 5-(4-Carboxy-
phenyl)-10,15,20-triphenyl-
1H,23H-porphyrin was purchased from Frontier Scientific Inc.,
anoma B78-H1 cells, with an IC50 value of about 800 nm,
whereas they showed no cytotoxicity in the dark.
2
Logan, UT, USA, and the N-hydroxysuccinimidyl ester (NHS ester) of
12-azido-4,7,10-trioxadodecanoic acid was obtained from Cyana-
gen. All other chemicals were obtained from Sigma–Aldrich and
were of the highest purity commercially available. TLC analysis was
conducted on TLC plastic sheets 60 F254 (Fluka) with detection by
UV absorption where applicable and/or by staining with a solution
of ammonium molybdate and/or a solution of ninhydrin, followed
by charring at approximately 1508C. Column chromatography was
performed using silica gel (0.063–0.200 mm particle size, 70–
The bioactivity of SPION-TPP nanoparticles is comparable to
that of TPP alone. As anticipated from the singlet-oxygen gen-
eration test, the conjugation of TPP to the SPIONs did not alter
1
the O production of unbound TPP. Unfortunately, the SPION-
2
TPP nanoconjugate had poor cell penetrability, which affected
the linearity of the dose–response effect. Enhanced cell deliv-
ery was achieved by conjugating a well-known cell-penetrating
peptide (HIV TAT peptide) to the SPION-TPP nanoparticles. The
Rhod-TAT-SPION-TPP nanoconjugates showed an improved IC50
value of about 500 nm and a linear dose–response effect. Inter-
2
30 mesh) purchased from Merck. Mass spectra were recorded on
an Applied Biosystems/MSD SCIEX QSTAR Hybrid Q-TOF mass spec-
trometer. H NMR spectra were acquired with a Bruker 200 NMR
1
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spectrometer (200 MHz). Chemical shifts are reported in ppm with
tetramethylsilane (TMS) as an internal reference. The abbreviations
used are: s=singlet, d=doublet, dd=doublet of doublets, m=
a final volume of 10 mL, and this was used as the stock solution of
SPION-N for further reactions.
3
1
General protocol for copper(I)-mediated “click” chemistry: Com-
pound 4 (2 mg; 2.4 mmol) was dissolved in 10 mm phosphate
buffer (500 mL; pH 7.5) (applying first 2 mL of DMSO). Sodium ascor-
bate (2 equiv.; 4.8 mmol; 0.9 mg) and CuSO ·5H O (0.5 equiv.;
multiplet, and br=broad. H-HRMAS-NMR experiments were per-
formed on a Bruker DMX 500 (11.7 T) equipped with an HRMAS
1
13
H– C indirect detection probe with gradients on the magic angle.
MAS experiments were performed at spinning rates up to 8 kHz
15 kHz maximum MAS rotation available) using a 50 mL zirconia
4
2
0
.3 mg; 1.2 mmol) were added to this solution. Finally, a solution of
(
SPION-N nanoparticles (500 mL) was also added and the reaction
3
rotor. In general, lyophilised nanoconjugate powder (1–2 mg) was
dispersed in deuterated DMSO (60 mL). Proton spectra were ob-
tained by using 1024 scans for each experiment. The sample tem-
perature was kept constant at RT.
mixture was rotated overnight. The resulting solution was dialysed
(SpectraPor regenerated cellulose, 3.5 KDa molecular weight cut-
off) against 10 mm ethylenediamine tetraacetic acid (EDTA) three
times over 12 h, against Milli-Q water for three days, and finally
against PBS three times over 12 h as the last step of the process.
Synthesis of compound 2: 2-[2-(Boc-amino)ethoxy]ethoxyacetic
acid (dicyclohexylammonium salt) 1 (100 mg; 225 mmol) was
placed in a round-bottomed flask, and dry DCM (40 mL) was
Synthesis of Rhod-TAT: The detailed synthetic protocol is de-
scribed in the Supporting Information, and involves a slight varia-
added. EDC (1.2 equiv.; 270 mmol; 52 mg), HOBT·H O (1.2 equiv.;
2
[23]
tion of a synthesis published previously by our group.
2
70 mmol; 41 mg), and TEA (2 equiv.; 450 mmol; 63 mL) were added
to the solution, which was stirred for 2–3 min. Propargyl amine
1.2 equiv.; 270 mmol; 17 mL) was then added to the mixture, which
e Measurement by UV spectroscopy: A standard solution of 2 mm
TPP in DMSO was prepared. This solution (50 mL) was diluted in
(
was stirred overnight. The reaction was monitored by TLC (5%
MeOH in DCM). After completion of the reaction, the mixture was
evaporated to remove the solvent. The evaporated contents were
purified directly by column chromatography (3% MeOH in DCM)
without any prior separation. The pure product (57 mg) was ob-
0
.5, 1, 1.5, and 2 mL of PBS and the UV absorbance was measured.
The experimental value of e at 422 nm for TPP was obtained by
using the Beer–Lambert law, and was calculated to be 1.43ꢁ
1
5
À1 À1
5
0 cm
m
(SD=0.45ꢁ10 ) as an average of several measure-
ments.
1
tained as a colourless, viscous liquid in 89.23% yield. H NMR
(
3
7
200 MHz, CDCl , 208C): d=1.36 (s, 9H), 2.21 (t, 2H), 3.28 (dd, 2H),
.47–3.62 (m, 7H), 3.94 (s, 2H), 4.04–4.00 (dd, 2H), 4.99 (br s, 1H),
.17 ppm (br s, 1H).
UV/Vis measurements: UV/Vis spectra were measured on a Varian
Cary 50 spectrophotometer. Spectra were measured from 200 to
1000 nm, and PBS was used as the baseline (see Figure S1 in Sup-
porting Information). The TPP conjugation on SPIONs was checked
through UV/Vis spectroscopy. The Q-band changes were the most
significant. Two methods were used to estimate the concentration
of TPP loaded on the nanoparticles. One method was based on
3
Synthesis of compound 3: Compound 2 (9 mg) was placed in
a round-bottomed flask. A 1:1 solution (v/v) of TFA in DCM (10 mL)
was added, and the reaction mixture was allowed to stand for 2 h.
The solution was evaporated, and this was followed by co-evapora-
tion with toluene (three times) and co-evaporation with TEA (three
times). The contents were dissolved in DCM, evaporated, and dried
to ensure the removal of volatile components. The product was
obtained quantitatively as observed by TLC (5% MeOH in DCM)
and used directly for the next step.
the correction of the TPP-SPION absorbance (A ) by the
TPPÀSPION
SPION absorbance (ASPION). In this approach, the Beer–Lambert law
was applied to DA=A at lmax 422 nm (e=1.43ꢁ
ÀA
TPPÀSPION
SPION
5
À1
À1
1
0 m cm , see Figure S1D in the Supporting Information). The
[29]
second method was based on a designed deconvolution spread-
sheet. This procedure allows the complex absorbance spectrum of
TPP-SPIONs to be resolved into individual absorption bands. The
peak ascribable to the TPP loaded on SPIONs was calculated direct-
ly, and its absorbance value was used to calculate the concentra-
tion (see Figure S1A, B, and C in Supporting Information; details of
the procedure are also reported there). The concentration values
obtained with the two approaches were in good agreement.
Synthesis of compound 4: 5-(4-Carboxyphenyl)-10,15,20-triphenyl-
2
4
solved in dry DCM. TEA (2.0 equiv.; 30 mmol; 4.2 mL) was added to
this solution, which was left to stand for a few minutes. A solution
of 3 (1.5 equiv.; 23 mmol; 4.5 mg) dissolved in dry DCM was added,
and the reaction mixture was stirred overnight at room tempera-
ture. The resulting product was evaporated and purified using
1H,23H-porphyrin (10 mg; 15 mmol), EDC (1.5 equiv.; 23 mmol;
.4 mg), and HOBT·H O (1.5 equiv.; 23 mmol; 3.5 mg) were dis-
2
1
Generation of singlet oxygen: O production was evaluated by
2
measuring the time-dependent decay absorbance of the ADPA
maximum at 400 nm. A solution of 5-(4-carboxyphenyl)-10,15,20-
triphenyl-21H,23H-porphyrin (0.5 mm) containing ADPA (73 mm;
total volume 1.2 mL), and a solution of SPION-TPP (0.5 mm) also
containing ADPA (73 mm; total volume 1.2 mL) were prepared. A
solution of ADPA (73 mm) in PBS (total volume 1.2 mL) was also
prepared as a control. All solutions were placed in 24-well plates
and irradiated with a Philips halogen bulb (50 W, 12 V).
column chromatography (4% MeOH in DCM) to obtain 12 mg of
pure product. Yield: 94%; H NMR (200 MHz, CDCl , 208C): d=
1
3
À2.79 (s, 2H), 2.26 (t, 1H), 3.60 (dd, 2H), 3.7–3.67 (m, 6H), 4.10 (s,
2
(
7
6
H), 4.16–4.12 (dd, 2H), 7.00 (br s, 2H), 7.77 (m, 10H), 8.15–8.36
overlapping signals, 10H) 8.90–8.77 ppm (overlapping signals,
+
H); molecular weight of 4: 840.34, m/z=841.64 [M+1] , m/z=
+
2+
86.53 [M-2 phenyl+1] , m/z=421.82 [(M+2)] , m/z=342.76
2
+
[
M-2 phenyl+2]
.
General preparation for the biological experiments: B78-H1 ame-
lanotic murine melanoma cells were cultured in DMEM (low glu-
cose), which contained 10% fetal calf serum and antibiotics (Peni-
cillin 100 UmL , Streptomycin 100 mgmL , and Glutamine 2 mm,
purchased from CELBIO, Milan, Italy). 5-(4-Carboxyphenyl)-10,15,20-
triphenyl-21H,23H-porphyrin, as a positive control, was dissolved
in DMSO and conserved in aliquots of 0.5 mm at À208C. SPION-
TPP and Rhod-TAT-SPION-TPP solutions obtained from dialysis were
used as such and diluted in culture medium to the desired concen-
Synthesis of SPION-N nanoparticles: The NHS ester of 12-azido-
3
4
,7,10-trioxadodecanoic acid (5 mg, 15 mmol) and DIPEA (10 mL)
À1
À1
were added to a solution of SPION-NH nanoparticles (Nanomagꢂ-
2
D-spio NH surface; 2 mL) in 2 mL of 0.1m citrate buffer (pH 7.4).
2
The mixture was rotated overnight and then dialysed (SpectraPor
regenerated cellulose, 3.5 KDa molecular weight cut-off) against
pure water for three to five days to provide SPION-N nanoparti-
3
cles. The dialysed contents were diluted with deionised water to
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trations. Commercially available SPION-NH2 nanoparticles were
used as negative controls and diluted to a final concentration of
Fe comparable to those in the SPION-TPP and Rhod-TAT-SPION-TPP
samples.
Candiota, and Prof. Dr. Carles Arffls (Universitat Autꢀnoma de
Barcelona (UAB), Cerdanyola del Vall›s, Spain) for their assistance
with MRI measurements. Dr. Valentina Mollo and Prof. Dr. Paolo
Netti (Italian Institute of Technology@CRIB, Center for Advanced
Biomaterials for Health Care, Naples, Italy) are acknowledged for
the help with the TEM experiments. This work was supported by
Cell metabolism assay: B78-H1 cells were seeded in a 96-well
3
plate at a density of 5ꢁ10 cells/well in 100 mL of culture medium.
The following day, the SPION-TPP or Rhod-TAT-SPION-TPP nanopar-
ticles were added at different concentrations, and the cells were in-
“
TALENTS for an International House” (co-funded Marie Curie
action of AREA Science Park, Trieste, Italy) granted to S.C.; the
work done in Barcelona was funded by CICYT (CTQ2012–30930).
cubated in the dark for 3 h at 378C and 5% CO . This was followed
2
À2
by irradiation with a white halogen lamp (400 Wffm , XEF-152S;
San-Ei Electric Co., Ltd) for 30 min (14 Jcm ). A corresponding ex-
À2
periment was performed with treated cells and without light acti-
vation. After incubation for 24 h, the cell metabolic activity was
evaluated through the resazurin assay, following the manufactur-
er’s instructions (Sigma–Aldrich, Milan, Italy). Data were obtained
with a spectrofluorometer Spectra Max Gemini XS (Molecular Devi-
ces, Sunnyvale, CA 94089).
Keywords: click chemistry
photodynamic therapy · porphyrin · superparamagnetic iron
oxide nanoparticles
·
magnetic properties
·
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Received: July 29, 2013
Published online on October 23, 2013
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