SWCNT-porphyrin nano-hybrids selectively activated by ultrasound: an interesting model for sonodynamic applications

Article abstract of DOI:10.1039/d0ra03944f

Sonodynamic therapy (SDT) is an innovative anticancer approach, based on the excitation of a given molecule (usually a porphyrin) by inertial acoustic cavitation that leads to cell deathviathe production of reactive oxygen species (ROS). This study aims t

Full text of DOI:10.1039/d0ra03944f

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SWCNT–porphyrin nano-hybrids selectively
activated by ultrasound: an interesting model for
Cite this: RSC Adv., 2020, 10, 21736
sonodynamic applications†
a
b
a
a
Federica Bosca,‡ Ingrid Corazzari,‡ Federica Foglietta, Roberto Canaparo,
c
de
a
a
a
Gianni Durando, Linda Pastero,
Silvia Arpicco, Franco Dosio, Daniele Zonari,
a
a
a
be
Giancarlo Cravotto,
and Alessandro Barge
Silvia Tagliapietra, Loredana Serpe, Francesco Turci
a
*
Sonodynamic therapy (SDT) is an innovative anticancer approach, based on the excitation of a given
molecule (usually a porphyrin) by inertial acoustic cavitation that leads to cell death via the production of
reactive oxygen species (ROS). This study aims to prepare and characterize nanosystems based on
porphyrin grafted carbon nanotubes (SWCNTs), to understand some aspects of the mechanisms behind
the SDT phenomenon. Three different porphyrins have been covalently linked to SWCNTs using either
Diels–Alder or 1,3-dipolar cycloadditions. ROS production and cell viability have been evaluated upon
ultrasound irradiation. Despite the low porphyrin content linked on the SWCNT, these systems have
shown high ROS production and high tumour-cell-killing ability. The existence of a PET (photoinduced
electron transfer)-like process would appear to be able to explain these observations. Moreover, the
demonstrated ability to absorb light limits the impact of side effects due to light-excitation.
Received 1st May 2020
Accepted 29th May 2020
DOI: 10.1039/d0ra03944f
rsc.li/rsc-advances
sonosensitizer molecules (usually a porphyrin ring) which, in
turn, induces the generation of ROS (including singlet oxygen,
Introduction
Sonodynamic therapy (SDT) was introduced as an innovative and hydroxyl radicals). On the other hand, the sonosensitizer
1,2
anticancer approach in 1989. Three main players are involved excitation could be directly produced by the high punctual
in this treatment, namely: (i) low-intensity ultrasound (US) used temperature and pressure conditions induced by cavitation,
as the energy source, (ii) a chemical sonosensitizer that is following a thermal energy transfer mechanism. Moreover,
activated by US induces the production of reactive oxygen direct radical production from water or from the sonosensitizer
7
–9
species (ROS), and (iii) the ROS themselves, which damage and itself could also be implied. Porphyrin derivatives, which are
3
10
kill target cells. The molecular mechanism of SDT is still non-toxic to cell culture per se are considered as the rst-
4
unknown, although several hypotheses have been proposed.
generation sensitizers and extensively used as model
Sonoluminescence as well as sonochemical and physical compounds in photo- and sono-dynamic treatment.
effects (namely high pressure and temperature), caused by the However, in the clinical practice prolonged skin photosensi-
11,12
1
13
implosion of cavitation gas bubbles (namely inertial cavitation), tivity aer systemic administration of porphyrins is observed.
are thought to be mainly involved. Sonoluminescence is This side-effect represents one of the major shortcoming in the
5,6
14
a process, invoked in a controversial way, in which gaseous clinical use of such sensitizers. To overcome this problem it is
species dissolved in solution are excited by acoustic cavitation possible to select sonosensitizer with different structure, or to
and become able to emit light. The light emitted would excite select molecules able to be excited only by US. Another strategy
envisages the enhancement of the sonosensitizer ability to
generate ROS. This approach allows to reduce the dose and,
a
consequently, the sonosensitizer concentration in the skin.
Furthermore, the sonosensitizer may also be made tissue
selective by conjugating it to a specic vector or including it in
targeted nanoparticles. To conjugate sonosensitizer to vector,
more than one strategy can be simultaneously followed. Single
walled carbon nanotubes (SWCNT), are one of the most prom-
Department of Drug Science and Technology, University of Turin, Turin, Italy. E-mail:
alessandro.barge@unito.it
b
Department of Chemistry, University of Turin, Turin, Italy
c
National Institute of Metrological Research (INRIM), Turin, Italy
d
Department of Earth Sciences, University of Turin, Turin, Italy
e
“
G. Scansetti” Interdepartmental Centre, University of Turin, Via P. Giuria 9, 10125
Turin, Italy
15,16
ising materials in nanomedicine
and they represent an ideal
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
model to build a new kind of porphyrin based sonosensitizer
0.1039/d0ra03944f
First author.
(porphyrin–SWCNT conjugated). SWCNT are known for their
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17,18
use in drug delivery and targeted drug delivery,
but they are of meso-tetrakis-(carboxyphenyl)-porphyrin with a-Boc-lysine t-
also known for their peculiar dimensional features (micro- butylester in presence of ethyl-diisopropylcarbodiimide (EDC)
metric dimension along z axis, and stiffness) and electronic and 4-N,N-dimethylaminopyridine (DMAP).
19–21
properties.
Three main advantages are expected from the
Porphyrins 1 and 3 expose furanyl terminal groups, which
porphyrin–SWCNT nanohybrid: (i) as described by Baskaran are appropriate for Diels–Alder (DA) cycloadditions. Porphyrin 2
22
and co-workers, the stiffness and micrometric size of SWCNT was synthetized to employ the a-aminocarboxylic moiety as
bundle favour the inertial acoustic cavitation to occur in the precursor of azomethine ylides, which is exploitable in 1,3-
proximity of the carbon surface where the sonosensitizing agent dipolar cycloadditions. All graing reactions with porphyrin 1, 2
23
is linked, (ii) the exceptional electron mobility of SWCNT
and 3, were performed by mono-modal microwave enhanced
assists the electron transfer between covalently bonded mole- cycloaddition. The functionalization degree of these three
cules and the nanotube, imparting a US-harvesting antennae hybrid materials was assessed by thermogravimetric analysis
2
0,22,24–26
behaviour
and (iii) SWCNT allows a facile chemical (Fig. S7, ESI†).
16
graing of porphyrin derivatives onto the carbon surface and
allow further functionalization with target agents.
study three different porphyrin–SWCNT nanohybrids were were detected, namely at 25–160 C (region I) and 160–270 C
The thermal degradation of CNT–porphyrin conjugates was
15,17,27,28
ꢀ
In this followed in the temperature range 270–700 C. Three regions
ꢀ
ꢀ
synthesized and studied in in vitro sonodynamic applications.
(region II) the desorption of molecular water and traces of
solvent deriving from the synthesis (N,N-dimethylformamide,
DMF) accounts for a minor weight loss (<3%). The weight loss at
this temperature (270 C) was used as reference point (offset) to
calculate the amount of porphyrin of each conjugate. The
ꢀ
Results and discussion
To assess if the ability in amplifying/facilitating the inertial
cavitation conditions and the electron transfer phenomenon
from porphyrin to SWCNT may increase toxic effect on cancer
cells, we conjugated the SWCNT surface with three ad hoc
tailored porphyrin derivatives. The three nanohybrid systems
amount of porphyrin was quantied by subtracting the offset of
ꢀ
ꢀ
each curve at 270 C from the weight loss at 700 C, in which the
thermal degradation of porphyrin is completed. The percentage
of porphyrin per mass unit of conjugate (SWCNT + porphyrin)
introduced by the functionalization process in SWCNT–1,
SWCNT–2 and SWCNT–3 was 6.7% w/w, 8.0% w/w and 7.0% w/
w, respectively. To obtain the functionalization degree (i.e., the
molar concentration of porphyrin per mass unit of conjugate)
the wt% were converted by using the molar mass of each
porphyrin. The functionalization degree of SWCNT–1, SWCNT–
(namely SWCNT–1, -SWCNT–2 and SWCNT–3), obtained by
graing these porphyrins onto SWCNT surface (Fig. 1), differ in
linking modality and consequently in spatial relative disposition.
Porphyrins derivatives 1 and 3 were obtained as previously re-
29,30
ported,
whereas derivative 2 was synthesized via condensation
2, and SWCNT–3 reported as mmol of porphyrin per mg of
sample was calculated as follows: functionalization degree ¼
3
(
(
D
700–270
ꢀ
C
(mass loss%)/MW porphyrin) ꢁ 10 . This result
Table 1) evidenced that similar functionalization degrees were
obtained in all cases, highlighting that the different graing
reactions, as well as the distance between furanyl moiety and
porphyrin core, do not signicantly affect the graing yield,
ꢂ2
ꢂ1
which ranged in all cases form 4.3 ꢁ 10 to 1.2 ꢁ 10 mmol of
porphyrin per mg of sample.
With the purpose to highlight a possible role of porphyrin
linking modality, the toxic effect of these hybrid nanosystems
on tumour cells was then evaluated. To proceed with cellular
studies, it was necessary to stabilize the aqueous suspension of
the modied SWCNT, reducing the aggregation phenomena.
The three SWCNT–porphyrin hybrids were wrapped with PEG
Table 1 The functionalization degree of SWCNT–1, SWCNT–2, and
SWCNT–3 reported as mmol of porphyrin per mg of sample was
calculated as follows: functionalization degree ¼ (D700–270
ꢀ
C
(mass
3
loss%)/MW porph) ꢁ 10
Porphyrin MW
Functionalization
Porphyrin wt% degree (mmol mg )
ꢂ1
ꢂ1
Nano-hybrid (mg mmol
)
ꢂ1
ꢂ2
ꢂ2
SWCNT–1
Structures of synthesized porphyrins and 3D pictures of SWCNT–2
574
1411
1634
6.7%
8.0%
7.0%
1.2 ꢁ 10
5.7 ꢁ 10
4.3 ꢁ 10
Fig.
1
porphyrins SWCNTs grafted.
SWCNT–3
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(
(
PEG ¼ polyethylene glycol) chains by using mPEG-DSPE
mPEG-DSPE 1,2-distearoyl-sn-glycero-3-phosphoethanol-
¼
amine-N-[amino(polyethylene glycol)-2000] ammonium salt)
31
under mild conditions. The attachment of PEG was conrmed
by thermogravimetric analysis (Fig. S6, ESI†), which reveals the
presence of one mPEG-DSPE chain each 104 carbon atoms
(weight loss of 69%, 250 mmol per g of SWCNT).
The hydrophobic lipid chains of the DSPE moiety interact
with the lipophilic carbon surface, decorating the SWCNT with
a PEG sunburst pattern and increasing the stability of SWCNT
aqueous suspension up to 30 days (Fig. S5, ESI†). A human
colorectal cancer cell line (HT-29), a well-known cellular model,
was then selected for in vitro tests. Cells were incubated with
a suspension of SWCNT–porphyrin–PEG (porphyrin: 1, 2 or 3) or
SWCNT–PEG (negative control) and exposed to US irradiation.
The results were compared to those obtained employing light
irradiation (since porphyrin is activable also by light), following
11,21,24
previously published procedures.
Cell proliferation at 24
and 48 hours aer each treatment was assessed (Fig. 3). The
incubation with SWCNT–1–PEG, SWCNT–2-PEG, and SWCNT–
3–PEG under US irradiation elicited a signicant reduction of
the cellular growth over time (up to 75%). Cell viability upon US
irradiation was not affected by the mode of conjugation, since
all the three different graing procedures gave very similar and
very low substitution degree and the differences shown in Fig. 3,
event aer 48 h of incubation, were not statistically relevant.
Fig. 3 Effect of the exposure of porphyrin-loaded SWCNT to LB and
US on HT-29 cell proliferation. HT-29 cells were exposed to
Light irradiation of functionalized SWCNT and non- porphyrin-loaded SWCNT–PEG (SWCNT–1–PEG; SWCNT–2–PEG
ꢂ1
functionalized SWCNT under both light- and US-irradiation and SWCNT–3–PEG, at 25mg ml ) and SWCNT–PEG and then either
ꢂ2
ꢂ2
LB (15 mW cm for 5 minutes, 405 nm) or US (0.008 mJ cm for
min, 1.866 MHz). Cell proliferation was evaluated after 24 and 48 h by
did not induce any signicant alteration in cell proliferation.
It is known that not conjugated porphyrins having similar
chemical structure to those used in this study are able to
5
WST-1 assay. Statistical significance between no exposure (full bars)
and LB or US exposure (dashed bars): **p < 0.01, ***p < 0.001.
32–34
generate ROS when light irradiated and kill cells
However,
in our experimental conditions, the lack of effect with light
irradiation is likely due to the absorption of most of the
impinging photons by the large black SWCNT surface, which,
combined with the modest amount of porphyrin graed,
prevents photoexcitation. To clarify the chemical mechanism at
the basis of the observed cell effect, reactive oxygen species
3
5
assessed by employing, terephthalate assay. US irradiated
porphyrin promotes ROS production with consequent genera-
tion of hydroxyl radicals which convert terephthalate into its

uorescent
derivative
2-hydroxyterephthalate
(TA-OH).
SWCNT–1 in DMSO : water (1 : 3) suspension was subjected to
US irradiation in the dark. The experiments were carried out
under two different US irradiation conditions: 1.866 MHz and
(ROS) generation by porphyrin loaded SWCNT hybrid was
22 kHz. The former was selected in order to match the experi-
mental condition used in cell treatments and the latter to test
2
our system in well-studied ROS production conditions. ROS
production of SWCNT–1 was compared with pristine tube
(SWCNT) under the same experimental conditions adopted in
cell experiments. To take into account ROS production via water
sonolysis, an aqueous solution of TA was irradiated under the
same conditions without SWCNT (blank + US). All the results
were also compared to a solution not subjected to sonication
(blank), to consider TA autoxidation. Under both US frequen-
cies, the ROS generated by SWCNT–1 were signicantly higher
than those observed with pristine SWCNT (p < 0.05). Notably,
pristine SWCNT did not induce ROS production more than
blank solution, indicating that the radical generation by our
hybrid nanomaterial relays on the occurrence of porphyrin
bonded to SWCNT surface.
Fig. 2 AFM and TEM images of SWCNT–1 (a–c) and pristine SWCNT
d–f).
(
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The results explain that cell death is a consequence of solely mediated ROS generation, representing the pivotal step of
US treatment, whereas light irradiation does not provide any sonodynamic applications.
effect on cell vitality. This phenomenon could be ascribed to the
Since the synthetic procedures share the same pressure and
low amount of porphyrin graed on the tube surface, which is temperature conditions, same type of reaction mechanism (i.e.
enough to promote a remarkable US-induced cellular toxicity, pericyclic reaction), produces very small amount of porphyrin
but insufficient to be excited by light (photons are mainly graed on SWCNT and, especially, the three nanosystems show
absorbed by SWCNT surface). SWCNT–porphyrin behaves as very similar biological behaviour, the structural and morpho-
a selective US-activated hybrid nanosystem. The thermal energy, logical properties of these nanohybrids were assessed only on
coming from inertial cavitation, is efficiently transferred to the SWCNT–1, using several complementary physico-chemical
few porphyrin units linked to the nanotube surface, causing characterization techniques. The results were compared with
molecule excitation, then, similarly, to what happens in those obtained with pristine carbon nanotubes (SWCNT).
a photo-induced electron transfer (PET) process, the electron Atomic force microscopy (AFM) and transmission electron
transfer between excited porphyrin (electron-donator) and microscopy (TEM) images (Fig. 2) were used to assess the nano-
carbon nanotube aromatic surface (electron-acceptor) stabilises morphology of pristine and graed SWCNT. High electron-
the separated-charge state, increasing the probability of dense areas (dark spots in panels c and f, in Fig. 2) are consis-
molecular oxygen to react with the excited system and facili- tent with the presence of metal residues and amorphous carbon
23
tating the ROS production via US excitation (Fig. 4).
attached to SWCNT bundles. The smallest structures detected
This feature can be further highlighted by comparing cell by AFM were composed of about ten single nanotubes, which
toxicity induced by our SWCNT–porphyrin system with that were directly observed by HR-TEM (panel b and e) and showed
induced by-decorated polymeric core–shell nanoparticle a minimum width of 7 nm. No signicant morphological
24
(
PCNP), under the same experimental conditions. A 40-fold modications were induced by the graing procedure.
lower dose of porphyrin graed on SWCNT is sufficient to As previously observed on reduced graphene oxide graed
21
induce a similar cell toxicity. So far, various porphyrin nano- with porphyrin, Raman spectroscopy conrmed that the
carriers have been investigated due to their inherent ability to functionalization process does not signicantly modify SWCNT
improve the triggering effect US has on the sonosensitizing structure (Fig. 5). Raman spectroscopy of carbon nanotubes,
36,37
agent.
0
properties, such as the photoinduced electron transfer, making strong peaks centred at ca. 1580 cm and 2700 cm (G and G
However, CNT-based nanocarriers shows unique and crystalline graphite in general, is characterized by two
ꢂ1
ꢂ1
them an intriguing multifunctional hybrid nanoplatform suit- bands, respectively). The lowest frequency peak (G-band, aer
able for the development of the next generation of US respon- graphite) is a rst-order Raman-allowed feature originating
sive nanosystems. Moreover, the results reported herein are from the in-plane stretching of the C–C bond. The highest
38,39
0
supported by the work of Yumita et al.
highlighting the great frequency peak (G band) is usually assigned to a second order
potential of CNT-based nanosystems in exploiting the US- two-phonon feature of crystalline graphite and, generally, of all
2
sp -carbon systems. In SWCNT, the rolled structure of the tube
breaks the two-dimensional graphene symmetry and up to six
high-energy tangential modes are allowed for the G-band. Those
modes can be practically treated considering only two intense
+
superimposed peaks labelled G , for atomic displacements
ꢂ
along the tube axis, and G , for modes with atomic displace-
+
ment along the circumferential direction. The G frequency is
constant with diameter for metallic and semiconductor tubes,
ꢂ
while G can be used to infer about the strain affecting CNT
structure, including nanotube curvature. At lower Raman shi
ꢂ1
(
ca. 1350 cm ), a peak is observed in the presence of defects
2
that discontinue the otherwise perfect sp graphite structure.
This peak is conventionally named D-band, where D stands for
defect or disorder. D-band in SWCNT is commonly used to
discuss the disorder-induced features related to symmetry
breaking of the sp conguration of graphene sheet. At Raman
shi ranging from 160–280 cm radial breathing mode (RBM)
2
ꢂ1
are usually observed for SWCNT. Fig. 5 A shows representative
spectra obtained from bundles of SWCNT with a low aperture
Fig. 4 TA hydroxylation induced by SWCNT–1 sonicated at different
frequencies (1.8 MHz and 22 kHz). Pristine and porphyrin-conjugated number objective and a 532 nm laser excitation source. Raman
SWCNT (SWCNT and SWCNT–1, respectively) suspensions and TA only
solution (blank) were sonicated (+US) for 5 min and 1 min with an
ultrasound probe operated at 1.8 MHz and 22 kHz, respectively. Data
spectra were background subtracted with a second-order poly-
nomial tting baseline and normalized for the Raman intensity
of the G band. As expected, the most prominent feature of
are reported as mean ꢃ standard deviation of triplicated experiments.
ꢂ1
pristine SWCNT was the G-band at ca. 1580 cm showing the
Columns that do not share at least one letter are statistically different
+
ꢂ
ꢂ1
(
ANOVA, Tukey test p < 0.05).
two G and G components at 1583 and 1516 cm , respectively,
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Because absolute intensity measurement is a difficult task in
Raman spectroscopy, the normalized intensity ID/IG ratio was
used to quantitatively describe the amount of disorder (Fig. 5B)
eventually induced by conjugation. Bearing in mind that the D-
band is not sensitive to all kind of defects, the relative low
intensity of this band with respect to the G-band clearly indi-
cates that the pristine SWCNT has a low amount of defective
3
rings, i.e. sp carbon atoms, open-end tubes, and sidewall holes.
To take into account that the laser source may easily overheat
and damage the samples, thus altering the Raman intensity of
D-band, a set of spectra were recorded on the same spot and
tube bundles were observed to be stable under laser irradiation
for minutes or even longer (data not shown). All the conjugates
obtained showed a negligible increase of the relative intensity of
+
the D-band with respect to G -band (I
D
/I
G
ratio), exhibiting
ratios between 0.08 and 0.16. To comparative analyse this range
of values, one should consider that a defective SWCNT might
4
0
exhibit I /I ratio > 1.6. The relative intensity and the down-
D
ꢂ
G
shi of G -band is virtually the same for all samples, indicating
that conjugation-induced strain did not signicantly affect tube
curvature. Furthermore, the functionalization did not induce
ꢂ1
shis in the main features of RBM at 186 and 270 cm , further
conrming the stability of tube diameter over conjugation.
Overall, the Raman spectra of the pristine and the variously
characterized SWCNTs were superimposable and the structural
integrity of tubes aer functionalization further conrmed.
Experimental
Materials and methods
All reagents and solvents were purchased from Sigma-Aldrich
and used without further purication, meso-tetrakis(4-
carboxyphenyl)porphyrin was purchased from Frontier Scien-
tic, Pure HiPco single walled carbon nanotube was purchased
from Unidym. MW assisted reactions were carried out in
a monomodal reactor Monowave 300 (Anton Paar) equipped
with a monitoring system of temperature, pressure and power
input. The CombiFlash RfsTeledyne (ISCO) purication system
was used to purify all crude reactions on silica gel. Analytical
and preparative HPLC-MS was performed using a FractionLink
autopurication system (Waters) equipped with a 2996 photo
Fig. 5 Representative Raman (lex ¼ 532 nm) spectra (A) and relative
intensity of the D-band (B) of pristine (SWCNT) and variously conju-
gates nanohybrids (SWCNT–1, SWCNT–2 and SWCNT–3). Raman diode array detector and Micromass ZQ detector (ESCI hybrid
+
graphitic band, split in the two singularities (G , for atomic displace-
ionization source). NMR spectra were recorded on an Avance
ꢂ
ments along the tube axis, and G for modes with atomic displace-
ment along the circumferential direction), ring breathing modes (RBM),
300 or on an Avance 600 spectrometers (Bruker) operating
respectively at 7 T and 14 T, dissolving samples in proper
deuterated solvent. Chemical shis were referenced per
residual solvent signals. MS spectra were carried out using
electrospray ionization (ESI) or by atmospheric pressure
chemical ionization (APCI), in positive ion mode, on a Micro-
and disorder band (D) were observed in all spectra. For each spectrum,
+
the relative intensity of the disorder band with respect to G band (I
D
/
I
G
) was calculated.
0
ꢂ1
and the G -band at 2645 cm . The sharpness and moderate mass ZQ spectrometer (Waters).
ꢂ
downshi of the G band of the pristine SWCNT signals the
Thermogravimetric analysis: TGA analyses were carried out
ꢂ1
semiconducting nature of the tube used in this work. The D- under dynamic nitrogen atmosphere (35 ml min ) by a Pyris 1
ꢂ1
band is observed at 1326 cm . A qualitative comparison of ultra-micro-balance; the gases evolved during the heating ramp
the peak shapes and frequencies between pristine and conju- were continuously monitored by a Spectrum 100 FTIR spectro-
gated SWCNT evidenced that the structural features of the tube scope (Perkin Elmer). The gas evolved during the heating ramp
ꢂ1
were largely preserved during the porphyrin conjugation. was piped (gas ow 65 ml min ) via pressurized heated (280
ꢀ
C) transfer line (Redshi S.r.l.) and analysed continuously by
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the FTIR (Spectrum 100, Perkin Elmer), equipped with a ther-
Synthesis of compound (1). Compound (1) was synthesized
ꢀ
29
mostated (280 C) conventional gas cell. The spectra were accordingly to previously described procedure, developed by
ꢂ1
acquired in the 4000–600 cm wavenumber range with a reso- authors, in 2% global yield.
ꢂ1
1
lution of 0.4 cm and analysed with the Spectrum soware
H NMR (300 MHz, THF-d8, d 1.73 ppm) d 9.18 (s, 8H), 8.25
(
Perkin Elmer). Temperature-resolved infrared proles of the (br s, 4H), 7.39 (br s, 4H), 7.08 (br s, 4H), ꢂ2.55 (s, 2H); APCI-
+
+
identied volatiles evolved from samples were obtained from MS : m/z calcd for C H N O , 574.16; found 575.48 [M + H ].
3
6
22 4 4
the intensity of a representative peak of the investigated species.
TEM analyses: the morphology of the samples was inves- amino-6-(((benzyloxy)carbonyl)-amino)-hexanoate.
Synthesis of compound (a) (CBzLys-t-BuO): tert-butyl 2-
Lys-CBz
tigated in the nanometric range by means of a JEOL 3010-UHR (10 g, 35.7 mmol) was dissolved in terz-butyl acetate (170 ml)
transmission electron microscope (TEM) equipped with and HClO (70% in water, 42 mmol, 3.6 ml) was slowly added.
4
a LaB lament operated at 300 kV, beam current ¼ 114 mA The mixture was stirred at RT for 18 h, then water (155 ml) was
6
and equipped with a 2k ꢁ 2k pixels Gatan US1000 CCD added. The two phases were separated and the aqueous layer
camera. Micro-Raman spectroscopy: the laser beam of was washed with ethyl acetate (2 ꢁ 50 ml). The organic layers
a confocal Raman microscope (Horiba Jobin-Yvon HR800 and were then combined and washed with a 5% NaHCO solution
3
Olympus BX41 microscope) has been focused on a 1 mm ꢁ 1 (2ꢁ 100 ml), dried over sodium sulphate and solvent was
1
mm wide portion of the SC. A polarized solid-state Nd laser removed to obtain (a) in 94% yield (11.25 g). H NMR (300 MHz,
operating at a wavelength of 532.11 nm and power of 80 mW DMSO-d , d 2.50 ppm) d 7.32 (m, 5H), 7.19 (t, J ¼ 5.42 Hz, 1H),
6
ꢀ
and a CCD air-cooled detector operating at ꢂ70 C were used. 4.99 (s, 2H), 3.82 (t, J ¼ 5.95 Hz, 1H), 3.00 (m, 2H), 1.72 (m, 2H),
1
3
Calibration of the instruments was performed by measuring 1.50–1.15 (overlapped signals, 13H); C NMR (75 MHz, DMSO-
ꢂ1
the Stokes and anti-Stokes bands of the Si band at 520.7 cm
.
6
d , d 39.5 ppm) d 169.6, 156.8, 137.8, 129.0, 128.4, 128.3, 83.5,
Samples were placed on a polished stainless-steel slide and 80.2, 65.8, 52.9, 30.6, 29.4, 28.1, 22.0; ESI-MS : m/z calcd for
+
+
a 50ꢁ objective delivering a power of ca. 15 mW on the sample C H N O , 336.42; found 337.19 [M + H ].
1
8
28 2 4
was used. Spectra were acquired with a spectral resolution of
Synthesis of compound (b) (CBzLysBoc-tBu): tert-butyl 6-
ꢂ1
ca. 2 cm and an integration time spanning from 100 to 400 (((benzyloxy)carbonyl)-amino)-2-((tert-butoxycarbonyl)-amino)
seconds.
hexanoate. Compound (a) (1.0 g, 2.97 mmol) was added to
Atomic force microscopy (AFM): AFM height prole measure- 50 ml of CH Cl and heated under reux until complete
2
2
ments were performed using a DME SPM Microscope (DME Igloo) dissolution. The solution was allowed to reach ambient
equipped with a DS95-50E scanner (scan volume 50 ꢁ 50 ꢁ 5 mm). temperature and Na CO (630 mg, 5.95 mmol) was added.
2
3
The data were acquired using MikroMasch Ultrasharp NSC16/Si
3
N
4
2
Mixture was stirred for 1 h then a solution of BOC O (1 g, 4.58
Cr–Au back-coated cantilevers with typical resonance frequency 190 mmol) in CH Cl (20 ml) was slowly added. The reaction pro-
2
2
ꢂ1
kHz, force constant 45 N m , tip radius lower than 35 nm and full ceeded under stirring overnight, then it was ltered ad washed
ꢀ
tip cone angle 40 . Due to the soness of the samples, all with 10% NaHCO solution (3 ꢁ 20 ml) and with water (2 ꢁ 20
3
measurements were performed in alternated contact mode.
ml). Organic phase was dried over sodium sulphate; solvent
Light exposure apparatus: a custom system for LB exposure was removed under reduced pressure and compound (b) was
with xed source-tube positioning and light diffuser all-around collected in 87.5% yield (1.13 g).
1
cell culture was used. The light-emitting source of the system is
3
H NMR (300 MHz, CDCl , d 7.26 ppm) d 7.19 (s, 5H), 4.94 (s,
based on InGaN light-emitting diodes (Cree Inc.) with 20 mW 2H + 2H), 3.99 (sb, 1H), 3.02 (sb, 2H), 1.73–0.96 (m, 9H + 9H +
1
3
max radiant power at a central wavelength of 405 nm. The 6H); C NMR (75 MHz, CDCl3, d 77.2 ppm) d 171.8, 156.5,
energy uency rates of the light radiation were adjusted to 15 155.5, 146.7, 128.4, 128.0, 127.9, 85.0, 81.6, 79.5, 66.4, 53.7,
ꢂ2
+
mW cm for 5 min, measured using Actinic UV-meter (Jelosil). 32.42, 31.1, 28.3, 27.9, 22.3; ESI-MS : m/z calcd for C H N O ,
2
3
36 2 6
+
US exposure apparatus: the US eld was generated by a plane 436.26; found 459.20 [M + Na ].
wave transducer in continuous wave at f ¼ 1.866 MHz connected to
Synthesis of compound (c) (LysBoc-tBu): tert-butyl 6-amino-
0
a power amplier (Type AR 100 A250A; Amplier Research, Sou- 2-((tert-butoxycarbonyl)-amino) hexanoate. Compound (b) (5 g,
derton, USA) and a function generator (Type 33250; Agilent). A 11.5 mmol) was dissolved in methanol (100 ml) and Pd/C (10%,
mechanical adaptor was built to connect the 1 cm diameter poly- 500 mg) was added. The mixture was stirred at room tempera-
styrene tube containing the cells suspended in PBS. When lled with ture under hydrogen atmosphere (atmospheric pressure) for
ultrapure water, the adaptor creates highly reproducible measure- 48 h. Then, it was ltered and solvent was removed under
ment conditions at a xed cell tube distance from the transducer.
reduced pressure obtaining a white solid (2.4 g). Acidimetric
Cell viability was evaluated by absorbance measurements titration indicates that this solid was formed by compound (c)
(
450 and 620 nm as reference wavelength) on a microplate (40%) and its carbamic acid form (60%). The solid was dissolved
ꢀ
reader (Asys UV340, Biochrom).
in methanol, cooled down to 5 C in an ice bath, and aqueous
2
HCl (1 M, 4.6 ml) was slowly added. The CO was removed under
vacuum, NaOH (2 M) was added until pH ¼ 9, methanol was
Synthetic procedures
evaporated under reduced pressure ad resulting aqueous phase
Compound 1 and 3 were synthesized accordingly with reported was extracted with ethyl acetate (3ꢁ 50 ml). Organic layer was
30
procedures. Whereas the procedure to obtain compound 2 is dried over sodium sulphate and solvent evaporated to obtain
here reported.
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compound (c) in 15% yield. Acidimetric titration indicates 85%
of purity.
Synthesis of SWCNT–2. SWCNT (30 mg), octanal (26 mg, 32
ml, 0.20 mmol), compound 2 (55 mg, 0.04 mmol) and DMF (3
1
H NMR (300 MHz, CDCl , d 7.26 ppm) § d 3.82 (sb, 1H), 2.48 ml) were placed in a specic glass tube (Anton Parr G10). The
3
+
ꢀ
(
sb, 2H), 1.82–0.70 (overlapped, 6H + 18H); ESI-MS : m/z calcd tube was placed in the MW oven, sealed and heated at 120 C
+
for C15
Synthesis of compound (2) meso-tetrakys-(4-(5-amino-5-car- W) for 2 h. The mixture was then cooled to RT, centrifuged and
boxypentylaminocarbonyl)-phenyl)-porphyrin.
Commercial washed with DMF (3 ml) and CH Cl (3ꢁ 4 ml). The solid was
meso-tetrakys-(4-carboxyphenyl)-porphyrin (64 mg, 0.08 mmol) recovered and dried at 100 C for 48 h. The nal product was
was dissolved in a mixture of CH Cl (30 ml) and THF (10 ml), characterized by Raman spectroscopy and TGA.
then EDC (165 mg, 0.86 mmol) and DMAP (52 mg, 0.43 mmol)
Synthesis of SWCNT–3. SWCNT (10 mg), compound 3
H
30
N
2
O
4
, 302.22; found 303.19 [M + H ].
(temperature-controlled MW irradiation with max power of 600
2
2
ꢀ
2
2
were added. The mixture was stirred at RT for 30 min and (10 mg, 6 mmol) and DMF (3 ml) were placed in a specic glass
a solution of compound (c) (306 mg, 85% purity, 0.86 mmol) in tube (Anton Parr G10). The tube was placed in the MW oven,
ꢀ
CH
monitored by TLC (silica, CH
Waters SunFire C18 column ꢂ4.6 ꢁ 150, 5 mm; ow ¼ 1 cooled to RT, centrifuged and washed with DMF (3 ml) and
2
Cl
2
(30 ml) was slowly dropped. The reaction progress was sealed and heated at 100 C (temperature-controlled MW irra-
2
Cl
2
/MeOH ¼ 9/1; R ¼ 0.87), HPLC diation with max power of 600 W) for 2 h. The mixture was then
f
(
ꢂ1
ml min ; eluent A ¼ 0.1% TFA in water, B ¼ 0.1% TFA in CH Cl (3ꢁ 4 ml). The black solid was recovered and dried at
2
2
ꢀ
MeOH, gradient – time, B%- 0, 75; 15, 75; 27.5, 100; 42.4, 100; 100 C for 48 h (14 mg). The nal product was characterized by
Rt ¼ 29.05 min) and ESI-MS. Aer 4 h the reaction was Raman spectroscopy and TGA.
completed and the mixture was separated by ash chromatog-
raphy (Silica cartridge 12 g, CH Cl /MeOH: column volume,
2
2
Preparation of PEG–SWCNTs
MeOH%- 0,0; 10,0; 12, 19.6; 15.3, 19.6; 17.3, 39.9; 21.3, 100; 25.3,
00) and the product containing fraction was further puried by
SWCNT (1 mg) were suspended in 2 ml of mPEG-DSPE solution
1
ꢂ1
(2.5 mg ml in PBS pH 7.4) and bath sonicated for 3 h. Finally,
preparative HPLC-MS (Waters SunFire C18 column ꢂ19 ꢁ 100,
unbound conjugate was removed by repeat ltering and
washing with PBS (Centrisart® I centrifugal ultraltration unit
NMWCO 100 kDa, Sigma-Aldrich).
ꢂ1
5
mm; ow ¼ 20 ml min ; eluent A ¼ water, B ¼ MeOH,
gradient – time, B%- 0, 75; 12.5, 100; 16, 100) obtaining 50 mg of
2) in 34% yield. The H-NMR spectrum shows the presence of
2) in mixture with its partially deprotected derivatives which do
1
(
(
1
mg of the different PEG-SWCNTs were added to 2 ml of
different medium [water, PBS, RPMI (Roswell Park Memorial
Institute medium) 1640 and RPMI 1640 + 10% FBS (foetal
bovine serum)] and bath sonicated for 1 h then stability within
not compromise its use in the next deprotection step. The solid
was then dissolved in CH Cl (20 ml) and TFA (3 ml) was slowly
2
2
added. The mixture was stirred overnight and solvent and
excess of TFA were evaporated under reduced pressure obtain-
30 days was evaluated. The suspension appears stable aer so
long period (Fig. 5,† in ESI,† shows the porphyrin–CNT–PEG
suspension aer 30 days from its preparation).
ing a blue/green solid (34 mg, yield 53%).
1
3
H NMR (300 MHz, CD OD, d 3.31 ppm) d 8.82 (s, 8H), 8.31
For the in vitro experiments PEG–SWCNTs were suspended in
(
(
m, 16H), 4.07 (sb, 4H), 3.62 (sb, 8H), 2.06 (sb, 8H), 2.29–1.5
ꢂ1
PBS containing 10% percoll® at a concentration of 500 mg ml
.
1
3
overlapped, 24H); C NMR (75 MHz, CD OD, d 49.0 ppm, from
3
+
HSQC) d 136.8, 131.4, 127.2, 53.5, 40.4, 30.8, 29.9, 23.2; ESI-MS :
m/z calcd for C72H N O12, 1302.59; found 1304.0 [M + H ],
78 12
6
+
Cell culture and treatments
+
52.6 [M + 2H ].
Human colorectal cancer cell line, HT-29 (ICLC, Interlab Cell
Synthesis of compound (3). Compound (3) was synthesized Line Collection, Genova, Italy), was cultured in RPMI-1640
3
0
ꢂ1
accordingly to previously described procedure, developed by medium, supplemented with 2 mM L-glutamine, 100 IU ml
1
ꢂ1
authors, in 46%. Yield (28.5 mg) H NMR (300 MHz, CD3OD, penicillin, 100 mg ml
streptomycin and 10% (v/v) heat-
d 3.31 ppm) d 8.50 (s, 8H), 8.28 (br. t, 4H), 7.96 (br. d, 4H), 7.36 inactivated fetal calf serum (Sigma-Aldrich) in a humidied
ꢀ
(
3
d, J ¼ 5.64 Hz, 8H), 7.31 (br. d, 4H) 6.33 (d, J ¼ 5.84 Hz, 8H), atmosphere of 5% CO air at 37 C.
2
.66–3.54 (br. m, 16H + 16H + 8H), 3.07 (br. s, 8H).
Cells in the exponential growth phase were harvested with
Synthesis of SWCNT–1. SWCNT (20 mg), compound 1 0.25% trypsin and normalized to 5.0 ꢁ 105 cells in 3 ml of
ꢂ1
(
40 mg, 0.07 mmol) and DMF (4 ml) were placed in a specic medium containing 10% of percoll® and 25 mg ml of SWCNT
glass tube (Anton Parr G10). The tube was placed in the MW and porphyrin-loaded SWCNT (SWCNT–1–PEG, SWCNT–2–PEG
ꢀ
oven, sealed and heated at 100 C (temperature-controlled MW and SWCNT–3–PEG) in polystyrene tubes for light beam (LB) or
irradiation with max power of 600 W) for 2 h. The mixture was US exposure.
then cooled to RT, centrifuged and washed with DMF (3 ml) and
LB exposure was carried on by custom system, adjusting
ꢂ2
CH Cl (3ꢁ 4 ml). The black solid was recovered and dried at energy uency rates of light radiation at 15 mW cm , for 5 min.
2
2
ꢀ
1
00 C for 48 h (21 mg). The nal product was characterized by The light intensity was measured using Actinic UV-meter
Raman spectroscopy, TGA, AFM and TEM.
(Jelosil, Le Landeron, CH).
US cells irradiation was carried out at f ¼ 1.866 MHz and
0
ꢂ2
with an energy density corresponding to 0.008 mJ cm ; energy
supplied to the cells did not increase the medium temperature
(maximum temperature recorded was 33 C).
§
Some impurities were observed; however chemical shis refer to most abundant
ꢀ
compound.
21742 | RSC Adv., 2020, 10, 21736–21744
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Cell proliferation assay
each synthetic route. Namely, the SWCNT conjugated via Diels–
Alder cycloaddition (compounds SWCNT–1 and SWCNT–3) and
azomethine ylide 1,3-dipolar cycloaddition (SWCNT–2) were
thoroughly characterized comparing pristine SWCNT.
The effect that porphyrin loaded SWCNT had on HT-29 cell
proliferation was evaluated by WST-1 cell proliferation assay
(
Roche Applied Science, Penzberg, Germany). Aer various
3
treatments, 2.0 ꢁ 10 HT-29 cells were seeded in 100 ml of
growth medium in replicates (n ¼ 8) in 96-well culture plates Conclusions
(
(
TPP, Trasadingen, Switzerland). At 24 and 48 h, WST-1 reagent
10 ml) was added and the plates were incubated at 37 C in 5%
ꢀ
In this study, we propose a novel covalently graed SWCNT–
porphyrin hybrid nanosystem, which is biologically active only
upon US irradiation thanks to the synergistic action of the two
bonded entities. The hybrid nanosystem is non-toxic per se in
vitro but kills up to 75% HT-29 colon cancer cell under low-
power US irradiation. The selectivity (no effect under LB) and
CO for 1.5 h. Well absorbance was measured at 450 and 620 nm
2
(reference wavelength) on a microplate reader Asys UV 340.
Statistical analyses
Data are shown as average values ꢃ standard deviation of three high efficiency (40-fold higher than PCNP) of these hybrid
independent experiments. Statistical analyses were performed nanosystems are here demonstrated for the rst time and it
on Graph-Pad Prism 6.0 soware (La Jolla, CA, USA); two-way might be due to the increase of the separated-charge state
analysis of variance and Bonferroni's test were used to calcu- lifetime, which promotes ROS production and, in turn, may
late the threshold of signicance. Statistical signicance was set impair cell proliferation. The lack of photo-induced effects of
at p < 0.05.
these SWCNT–porphyrin could allow to overcome the big limit
of skin photosensitivity. The here described hybrid nanosystem
represents an interesting model for a further development of
selective US-activated anticancer sonosensitizer.
Reactive oxygen species (ROS) generation/terephthalate assay
Terephthalate (TA) solution (8 mM) was prepared dissolving
disodium terephthalate in Milli-Q ultrapure water. A stable
SWCNT-1 suspension was obtained suspending SWCNT–1 in
DMSO (purity 99.9%). TA solution was added to the DMSO : -
water (1 : 3) suspension of SWCNT–1. The concentration of TA
and SWCNT–1 in the nal mixture was 4.0 mM, and 25 mg ml
Conflicts of interest
There are no conicts to declare.
ꢂ1
,
respectively. The suspension was introduced in a polystyrene Acknowledgements
tube (volume ¼ 3 ml; tube diameter ¼ 1 cm) and exposed to US
This research was supported by MIUR (fondi ricerca locale 2013
e 2014), by Associazione Italiana per la Ricerca sul Cancro (AIRC
(
1.866 MHz) under the same experimental conditions employed
ꢂ2
in cell experiments (namely 0.008 mJ cm for 5 min). Sonica-
–
MFAG 2012, MFAG-13048) and by Compagnia di San Paolo,
tion was carried out using also different sonotrode frequency
Torino Bando CSP 2015 (BiNGO project). The authors are
grateful to Prof. L. Anfossi for granting access to the FLx800
(
22 kHz): 2 ml of suspension were introduced in polystyrene
ꢂ2
tubes and exposed to ultrasound (10 kJ cm
for 1 min)

uorescence reader, and Dr A. Troia for his help during some
employing a sonicator Sonopuls HD3100 equipped with a 73 MS
probe (Bandelin, Germany). Aer the US treatment, the
suspension was ltered on a cellulose acetate membrane (pore
diameter 0.45 mm, Advantec, Japan) and TA-OH uorescence
irradiation experiments. The TGA-FTIR-GC-MS and micro-
Raman spectroscopy measurements were obtained with the
equipment acquired by the ‘G. Scansetti’ Interdepartmental
Center for Studies on Asbestos and Other Toxic Particulates,
thanks to a grant by the Compagnia di San Paolo, Torino, Italy.
(
l
ex ¼ 324 nm, lem ¼ ca. 425 nm) was measured on the clear
solution by a FLx800 uorescence reader (BioTek, US). The
results were compared to a blank solution without US irradia-
tion and on US-irradiated pristine SWCNT (25 mg ml ) under
ꢂ1
Notes and references
the same experimental conditions. Three replicates for each
experiment were carried out and the results were expressed as
the mean values ꢃ SD of the separate determinations and
analysed by a one-way Analysis of Variance (ANOVA) and
Tukey's test. p < 0.05 was considered signicant.
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21744 | RSC Adv., 2020, 10, 21736–21744
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