Near-infrared and metal-free tetra(butylamino)phthalocyanine nanoparticles for dual modal cancer phototherapy

Article abstract of DOI:10.1039/d0ra03898a

Synergistic phototherapy combining photodynamic therapy (PDT) and photothermal therapy (PTT) based on near-infrared (NIR) dyes using a single light source offers the opportunity to treat diseases at deep locations. In this study, we reported human serum albumin (HSA)-involving tetra(butylamino)phthalocyanine (Pc)-based nanomaterials of HSA-α-Pc and HSA-β-Pc as highly efficient dual-phototherapy agents, namely 1(4),8(11),15(18),22(25)-tetra(butylamino)phthalocyanine (α-Pc) and 2(3),9(10),16(17),23(24)-tetra(butylamino)phthalocyanine (β-Pc). Both HSA-α-Pc and HSA-β-Pc showed excellent photothermal effects under a single NIR (808 nm) laser irradiation due to the S1 fluorescence emission quenching of Pcs. Compared to HSA-β-Pc, HSA-α-Pc exhibited better singlet oxygen generation ability and its highly efficient PDT/PTT dual-phototherapy was also well evidenced via in vitro and vivo experiments under a single 808 nm laser irradiation. Overall, this approach would be viable for the fabrication of more new Pc-based metal-free nano agents for PDT/PTT synergistic phototherapy upon a single NIR light source.

Full text of DOI:10.1039/d0ra03898a

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PAPER
Near-infrared and metal-free tetra(butylamino)
phthalocyanine nanoparticles for dual modal
cancer phototherapy†
Cite this: RSC Adv., 2020, 10, 25958
a
a
a
a
Anqi Xue,b
*
Ying-Jie Wu,‡ Fan-Hong Lv,‡ Jing-Lan Kan, Qun Guan,
Quanbo Wang,b Yan-An Li
and Yu-Bin Dong
a
a
*
Synergistic phototherapy combining photodynamic therapy (PDT) and photothermal therapy (PTT) based
on near-infrared (NIR) dyes using a single light source offers the opportunity to treat diseases at deep
locations. In this study, we reported human serum albumin (HSA)-involving tetra(butylamino)
phthalocyanine (Pc)-based nanomaterials of HSA-a-Pc and HSA-b-Pc as highly efficient dual-
phototherapy agents, namely 1(4),8(11),15(18),22(25)-tetra(butylamino)phthalocyanine (a-Pc) and
2(3),9(10),16(17),23(24)-tetra(butylamino)phthalocyanine (b-Pc). Both HSA-a-Pc and HSA-b-Pc showed
excellent photothermal effects under a single NIR (808 nm) laser irradiation due to the S1 fluorescence
emission quenching of Pcs. Compared to HSA-b-Pc, HSA-a-Pc exhibited better singlet oxygen
generation ability and its highly efficient PDT/PTT dual-phototherapy was also well evidenced via in vitro
and vivo experiments under a single 808 nm laser irradiation. Overall, this approach would be viable for
the fabrication of more new Pc-based metal-free nano agents for PDT/PTT synergistic phototherapy
upon a single NIR light source.
Received 30th April 2020
Accepted 3rd July 2020
DOI: 10.1039/d0ra03898a
rsc.li/rsc-advances
encapsulation with multistep fabrication, low reagent loadings,
and dual laser irradiation.3 So, the facile fabrication of single
Introduction
laser triggered, especially single near-infrared (NIR) laser
induced PDT/PTT nano agents is very signicant and
imperative.
Phototherapy, including photodynamic therapy (PDT) and
photothermal therapy (PTT), is a widely recognized approach
for cancer treatment. PDT and PTT require light absorption and
photosensitizers to generate reactive oxygen species (ROS) and
heat to kill cancer cells, respectively.1 Different from PDT, PTT,
as an oxygen independent phototherapy approach, can not only
kill all the cancerous cells including hypoxic ones, but also
increase the intratumoral blood ow and enriches tumor
oxygenation for boosting the oxygen-elevated PDT.2 Therefore,
the synergistic use of PDT and PTT dual-phototherapy can
achieve optimized efficacy in treating almost all the malignant
solid tumors. So far, the most reported PDT/PTT nanomaterials
were constructed by combing typical organic photosensitizers
(e.g. porphyrin, phthalocyanine, BODIPY, etc) with inorganic
photothermal conversion agents (e.g. nano Au, carbon dots,
metal sulde, etc) through surface modication or
Phthalocyanines (Pcs) have been considered as a promising
class of PDT materials because of their suitable Qmax band
absorption (650–700 nm), high extinction coefficients, tunable
photophysical and photochemical properties via facile chemical
modications.4,5 For example, aluminium Pc (Photosens®,
Russia) has been approved for clinical use.6 In contrast, only
a handful of Pc-based PTT examples have been reported so far.
As it is known, the light-to-heat conversion efficiency of the Pc-
based photothermal agents could be improved by effectively
reducing or quenching their uorescence and intersystem
crossing by formation of the self-assembled Pc aggregation,7
synthesis of the paramagnetic metal (e.g. Cu2+, Fe3+, Co2+, Ni2+,
etc) involved Pc metal complexes, and tuning the peripheral
substituents (e.g. amino group through phenoxy as a bridge) or
axial ligands (e.g. amino group through phenyl as a bridge) of
Pcs.8 In some cases, Pcs not only serve as the ROS generation
agent but also as the light-to-heat conversion agent by self-
quenching excited states arising from the compactly packed
monomer, which might allow the synchronous PDT/PTT under
a single laser to be possible. For example, the hydrophobic Pc-
loaded silica NPs (Pc@HSNs) reported by Li and co-workers
exhibited efficient dual PDT and PTT effects,9 and Kim and
co-workers recently reported the nanostructured Pc-assemblies
aCollege of Chemistry, Chemical Engineering and Materials Science, Collaborative
Innovation Center of Functionalized Probes for Chemical Imaging in Universities of
Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education,
Shandong Normal University, Jinan 250014, P. R. China. E-mail: kanjinglan@163.
com; yubindong@sdnu.edu.cn
bShandong Analysis and Test Center, Qilu University of Technology (Shandong
Academy of Sciences), Jinan 250014, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra03898a
‡ Contributed equally.
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(PcTBs) which displayed intrinsically unique photothermal and
photoacoustic properties, but they all induced by the non-NIR
Illumination (l < 750 nm).10
Experimental
Materials
An ideal agent for PTT/PDT combinatorial therapy should
possess high photothermal conversion efficiency, meanwhile
exhibit strong NIR (l > 750 nm) absorption, which is a trans-
parency window for biological tissues.11,12 However, the Qmax
bands of the most Pc-based photothermal agents are located
within 650–700 nm. To date, only one example of Pc-
nanomaterial for NIR irradiated synchronous PDT/PTT was re-
ported, in which the metal-involved ZnPc nanowire (NW) was
DMF was distilled from CaH2. All other commercial solvents
and reagents were used without further purication unless
otherwise mentioned. 3/4-Nitrophthalonitrile, n-butylamine,
1,3-diphenylisobenzofuran (DPBF), Li, and extra dry n-butanol
were purchased from Energy Chemical Co., Ltd. Trimethyl-
amine was purchased from Sinopharm Chemical Reagent Co.,
Ltd. Human serum albumin (HSA) and 3-(4,5-dimethyl-2-thia-
zolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were
purchased from Sigma-Aldrich (Shanghai) Trading Co. Ltd.
Phosphate-Buffered Saline (PBS) and Dulbecco's Phosphate-
Buffered Saline (DPBS) were purchased from Biological Indus-
tries USA, Inc. RPMI Medium 1640 basic (1X) (RPMI 1640), Fetal
Bovine Serum (FBS), Penicillin–Streptomycin mixtures (Pen–
Strep) and trypsin–EDTA solution were purchased from
HyClone Laboratories, Inc. 20,70-Dichlorodihydrouorescein
Diacetate (DCFH-DA), Calcein Acetoxymethyl Ester (Calcein-
AM) and propidium iodide (PI) were purchased from
Shanghai Mackinlin Biochemical Co., Ltd. All organic solvents
were purchased from Sinopharm Chemical Reagent Co., Ltd.
Deionized water prepared with an Aquapro System (18 MU) was
employed in all experiments.
prepared by
a vaporization–condensation–recrystallization
(VCR) multistep synthesis at 550 ꢀC in Ar stream.13
Our interest is to develop a facile approach for the fabrication of
the metal-free Pc-based nano agents for dual modal cancer pho-
totherapy upon single NIR laser irradiation. Besides NIR, the
metal-free Pc-nanomaterials could completely avoid the toxicity
potentially caused by metal species. In this contribution, two
metal-free and butylamino group peripherally decorated NIR Pcs,
namely 1(4),8(11),15(18),22(25)-tetra(butylamino)phthalocyanine
(a-Pc) and 2(3),9(10),16(17),23(24)-tetra(butylamino)phthalocya-
nine (b-Pc), were reported. More importantly, their human serum
albumin (HSA)14 assisted nanostructured assemblies of HSA-a-Pc
and HSA-b-Pc were fabricated via a facile approach, and they all
showed excellent photothermal effects under a single NIR (808
nm) laser irradiation. As HSA is the most abundant protein in
plasma, it can serve as a versatile carrier for drug delivery.
Currently, a number of clinical relevant HSA-based therapeutics
have been approved by the Food and Drug Administration (FDA),
and many more are under active clinical investigation.15 When
being used to fabricate nano agents, it is unlikely to cause any
undesired interaction with other serum proteins for these HSA-
based nanomaterials. Thus, these nanomaterials with extremely
low systemic toxicity have been studied as a versatile platform for
diagnosis and precision therapy.15–17 For example, Abraxane
fabricated via hydrophobic interactions between HSA and pacli-
taxel, a paclitaxel albumin nanoparticle, has been approved by FDA
for treating various cancers.18 Of note, HSA-a-Pc displayed a better
ROS generation ability than that of HSA-b-Pc, and its highly effi-
cient PDT/PTT dual-phototherapy was well evidenced via in vitro
and vivo experiments under a single 808 nm laser irradiation
(Scheme 1).
Characterizations
1H NMR spectra were recorded on a Bruker AVANCE III HD
400 M spectrometer. Spectra were referenced internally using
the residual solvent resonance relative to SiMe4. MALDI-TOF
mass spectra were taken on a Bruker BIFLEX III with alpha-
cyano-4-hydroxycinnamic acid as matrix. Elemental micro-
analyses (EA) were performed with an Elementar Vario EL Cube
Elemental Analyzer. The UV-vis absorption spectra were recor-
ded with a UV-2600 Jingdao spectrophotometer. Fluorescence
spectra were obtained with FLS-920 Edinburgh Fluorescence
Spectrometer with a Xenon lamp. Transmission electron
microscopy (TEM) was recorded on a Hitachi HT7700. Electron
microscope at an operating voltage of 120 kV. Dynamic Light
Scattering (DLS) and zeta potentials were obtained on a Malvern
Zetasizer Nano ZS90 with water as solvent at 25 ꢀC. Confocal
uorescence imaging studies were performed with a TCS SP5
confocal laser scanning microscopy (Leica Co., Ltd. Germany)
with an objective lens (ꢁ20).
Singlet oxygen generation
Singlet oxygen generation measurement was carried out by
modied method using 1,3-diphenylisobenzofuran (DPBF) as
capture agent.19,20 62 mL of DPBF (DMF, 1.33 mM) and 2 mL of
HSA-a-Pc or HSA-b-Pc aqueous solution (20 mM Pc equiv.) were
mixed in a quartz cuvette and irradiated under a 808 nm laser
(1.5 W cmꢂ2). The absorbance intensity of DPBF at 420 nm was
recorded at 1 min intervals. The rate of singlet oxygen genera-
tion was determined from the reduced absorbance intensity
over time. For the control experiments, DPBF absorption was
also recorded for negative comparison at the same conditions in
the absence of photosensitizer.
Scheme 1 Schematic illustration of synthesis of HSA-a-Pc and HSA-
b-Pc NPs and their phototherapy effects.
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The cell culture medium was removed, and cells were washed
with ice-cold PBS buffer (pH ¼ 7.4) for two times before xed
with fresh 4.0% paraformaldehyde (1 mL) for 10 min at room
temperature. The xed cells were again washed with PBS 7.4 for
three times before observation by confocal laser scanning
microscope excited at 405 nm and monitored at 450–550 nm.
Photothermal activity
Aqueous solution (1.0 mL) of the Pc NP sample was put in
a quartz cuvette and irradiated with an 808 nm laser for 15 min.
Pure water was used as a control group. A thermocouple probe
with a digital thermometer was used to measure the tempera-
ture every 10 s. The photothermal conversion efficiency (h) was
calculated according to the reported method (eqn (1)–(3)):21,22
In vitro singlet oxygen generation tests
hAðTmax ꢂ TsurrÞ ꢂ QDis
h ¼
(1)
MCF-7 cells were incubated with samples (20 mM Pc equiv.) for
2 h, stained with 20,70-dichlorodihydrouorescein diacetate
(DCFH-DA) (20 mM) for 10 min and then washed with PBS 7.4.
Ið1 ꢂ 10ꢂA
Þ
l
P
miCi
hA ¼
(2)
(3)
Before and aer irradiation with an 808 nm laser (1.5 W cmꢂ2
)
ss
for 5 min, cell images were acquired using a confocal laser
scanning microscope. The ROS probe was excited at 488 nm and
monitored at 490–590 nm, laser intensity 20%, Smart Grain at
800 V.
t
ss ¼ ꢂ
ln q
where hA are the heat transfer coefficient and surface area of the
cuvette cell, respectively. Tsurr and Tmax are initial and nal
temperature of the solution. QDis represents the heat dissipa- In vitro phototherapy
tion of solvent (water), which was measured independently and
MCF-7 cells were incubated with samples (20 mM, Pc equiv.) for
found to be 0.014 W. I is incident laser power (1.18 W), and Al is
the absorbance at 808 nm. m and C are the mass (1 g) and heat
capacity (4.2 J gꢂ1) of water, respectively. ss is the time constant
of sample system. q is the dimensionless driving force and t is
time.
2 h and then irradiated with an 808 nm laser at a power density
of 1.5 W cmꢂ2 for 10 min. Aer 15 min incubation, both
Calcein-AM (calcein acetoxymethyl ester) and PI (propidium
iodide) were used to co-stain the cells to determine the photo-
therapy effect of the samples by a laser scanning confocal
microscope by using a green channel for live cells (lex ¼ 488 nm,
500–550 nm), and a red channel for dead cells (lex ¼ 543 nm,
600–650 nm).
Cell culture
Human breast adenocarcinoma cell line (MCF-7 cells) were
provided by Institute of Basic Medicine, Shandong Academy of
Medical Sciences (Jinan, China). MCF-7 cell lines were cultured
in RPMI 1640 supplemented with 10% (v/v) FBS and 1% anti-
biotics (penicillin/streptomycin, 100 U mLꢂ1) at 37 ꢀC under
a 5% CO2 atmosphere.
In vivo antitumor therapy
Nude mice (BALB/c-JGpt-Foxn1nu/Gpt, male, aged 5 weeks, ꢃ20
g) were purchased from the Beijing Vital River Laboratory
Animal Technology Co., Ltd. Animal experiments were reviewed
and approved by the Ethics Committee of Shandong Normal
University (Jinan, P. R. China). All the animal operations
complied with Chinese government relevant guidelines and
regulations for the care and use of experimental animals.
MCF-7 cancer cells (106 cells) suspended in DPBS (100 mL)
were subcutaneously injected into the anks of each mice to
establish MCF-7 xenogra model. Length (L) and width (W) of
the tumor were determined by digital calipers. The tumor
volume (V) was calculated by the formula V ¼ 1/2 ꢁ L ꢁ W2.
When the tumor size reached ꢃ150 mm3, the nude mice
bearing MCF-7 tumors (n ¼ 24) were randomly distributed into
6 groups. Aer intratumoral injection (200 mM, Pc equiv., 50
mL), the nude mice were feeding for 1 h, and for the treatment
group, light treatment (808 nm laser, 1.5 W cmꢂ2, 8 min) was
performed on the tumor site. The mice continued to be fed for
10 days. The tumor volume and nude mouse body weight were
recorded daily during the experimental period.
Cytotoxicity assay
The dark cytotoxicity of HSA-a-Pc and HSA-b-Pc NPs as well as
their photocytotoxicity were evaluated by 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were
plated at a density of 1 ꢁ 104/well in a 96-well cell culture plate
ꢀ
at 37 C under 5% CO2 atmosphere overnight. The cells were
treated with increasing concentrations (0, 5, 10, 20 mM Pc
equiv.) of HSA-a-Pc or HSA-b-Pc NPs (100 mL per well) as treat-
ment group, treated with free RPMI 1640 (100 mL per well) as
control group for 24 h at 37 ꢀC under a 5% CO2 atmosphere and
incubated for 2 h. Aer the removal of samples, cells were
transferred into fresh media and then irradiated by the 808 nm
laser at a power density of 1.5 W cmꢂ2 for 10 min. The dark
cytotoxicity (without irradiation) was monitored without irra-
diation at the same time as control. The cells were then incu-
bated at 37 ꢀC for additional 24 h before the MTT assay to
determine the cell viabilities.
Synthesis of 3/4-butylaminophthalonitrile
Confocal laser scanning microscopy (CLSM)
3/4-Nitrophthalonitrile (10.0 g, 57.8 mmol), n-butylamine (4.6 g,
The MCF-7 cells were seeded in glass-bottomed dishes in 2 mL 63 mmol), and triethylamine (11.7 g, 116 mmol) were dissolved
of RPMI 1640 medium. The cells were treated with samples in 50 mL dry DMF and stirred at room temperature for 12 h.
ꢀ
HSA-a-Pc or HSA-b-Pc NPs (both at 20 mM, Pc equiv.) at 37 C. Then, the mixture was poured into 500 mL water, and the
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precipitate was collected by ltration. The resulting precipitate 2.0 mL of the human serum albumin (HSA) aqueous solution
was recrystallized from dichloromethane and ethanol and gave (1 mg mLꢂ1) and stirred for 1 h. Aer repeating the above-
pure compound as an orange-yellow powder.
mentioned procedure for ve times, the HSA-a-Pc NP was ob-
3-Butylaminophthalonitrile. Yield, 89% (10.2 g). IR (KBr tained by centrifugation together and washed with water for
pellet cmꢂ1): 3374(vs), 3094(w), 2955(s), 2869(m), 2224(s), three times before storing in water (2.0 mL). 200 mL HSA-a-Pc
1956(w), 1860(w), 1763(w), 1613(vs), 1516(s), 1344(s), 1205(w), NP aqueous solution and 200 mL H2O were added to the mixture
1065(w), 775(s), 732(w), 571(w), 463(w). 1H NMR (400 MHz, solution of 200 mL DMSO and 2.0 mL chloroform and then 200
CDCl3, 25 ꢀC, TMS, ppm): 7.47–7.43 (t, 1H,–C6H3–), 7.01–6.99 (d, mL DMSO was added to the aqueous solution. Five minutes
1H,–C6H3–), 6.91–6.89 (d, 1H, –C6H3–), 4.81 (s, 1H, –NH–), 3.25– later, all the above mixtures were shaken until the phase of
3.21 (t, 2H, –CH2–), 1.70–1.63 (m, 2H, –CH2–), 1.48–1.43 (m, 2H, water changed from the dark-green to colorless. The concen-
–CH2–), 1.00–0.96 (t, 3H, –CH3).
trate of a-Pc in the above organic solution was assessed based
4-Butylaminophthalonitrile. Yield, 86% (9.8 g). IR (KBr on the UV standard working curves and then the amounts of a-
pellet cmꢂ1): 3363(vs), 3084(w), 2955(s), 2859(m), 2224(s), 1945(w), Pc in NPs aqueous solution could also be calculated. The HSA-b-
1623(vs), 1526(s), 1473(m), 1344(m), 1258(m), 1086(w), 850(s), Pc NP was prepared in the same way as that of HSA-a-Pc NP, in
1
ꢀ
732(w), 636(w), 539(m). H NMR (400 MHz, CDCl3, 25 C, TMS, which b-Pc was used instead of a-Pc. In the same way, the
ppm): 7.51–7.49 (d, 1H,–C6H3–), 6.83 (s, 1H,–C6H3–), 6.75–6.73 (d, amounts of b-Pc in NP aqueous solution used below was
1H, –C6H3–), 5.34 (s, 1H, –NH–), 3.19–3.16 (t, 2H, –CH2–), 1.68–1.60 assessed based on the UV standard working curves.
(m, 2H, –CH2–), 1.45–1.40 (m, 2H, –CH2–), 1.00–0.96 (t, 3H, –CH3).
Results and discussion
Synthesis and characterization of HSA-a-Pc and HSA-b-Pc NPs
Synthesis of metal-free tetra(butylamino)phthalocyanines
Li (28 mg, 4.0 mmol) in extra dry n-butanol (2.0 mL) was heated
As shown in Scheme 1, a-Pc and b-Pc were synthesized based on
to 90 ꢀC for 1 h under N2 atmosphere and then 3/4-
3-butylaminophthalonitrile and 4-butylaminophthalonitrile,
butylaminophthalonitrile (80 mg, 0.4 mmol) was added. The
respectively (for details see the ESI, Fig. S1–S3 and Table
resulting mixture was heated to reux for 4 h. Aer being cooled
S1†).23–25 The obtained a-Pc and b-Pc displayed a broad Q band
to room temperature, the volatiles were evaporated in vacuo and
at 786 and 741 nm, respectively (Fig. 1). Compared to their
the residue was chromatographed on a neutral alumina column
with dichloromethane : tetrahydrofuran (1 : 1) as eluent.
Repeated chromatography followed by recrystallization from
dichloromethane and methanol and gave pure tetra(butyla-
pristine H2Pc (Q band ¼ 672 nm),26 the large red-shis of 114
and 69 nm in their Q-band regions which was caused by the
strong electron-donating dibutylamino group and p–p conju-
gation between the N atom and the central phthalocyanine
chromophore. As is known, the prime window for the light
delivery through tissue is 700–900 nm, in which light absor-
mino)phthalocyanine compounds as dark powder.
1(4),8(11),15(18),22(25)-Tetra(butylamino)phthalocyanine
(a-Pc). Yield, 25% (20 mg). UV-vis [lmax (nm) (log(3), Mꢂ1 cmꢂ1)]:
bance and scattering from endogenous absorbers such as oxy-
CHCl3, 326 (4.83), 516 (4.10), 708 (4.68), 786 (5.08). IR (KBr
and deoxyhemoglobin, water and lipids are minimized.11
pellet cmꢂ1): 3309(w), 3078(w), 2963(vs), 2919(vs), 2574(w),
Therefore, both a-Pc and b-Pc with increased conjugation
length could be useful candidates to construct Pc-based nano-
materials for the NIR phototherapy.13
On the other hand, Pcs are known to be poor biocompati-
bility due to their notorious aggregating property. For
addressing such an issue, human serum albumin (HSA), which
has been demonstrated to be a useful drug carrier to sequester
1909(w), 1608(vs), 1510(s), 1466(m), 1341(vs), 1218(m), 1164(m),
1
1093(m), 1032(s). H NMR (400 MHz, CDCl3/[D6]DMSO (6 : 1),
ꢀ
25 C, TMS, ppm): 7.80–6.58 (–PcC H –), 3.64–3.38 (4H, –NH–),
6
3
2.20–1.78 (–CH2–), 1.18–0.79 (–CH2CH2CH3), ꢂ1.95 (–PcNH–).
MALDI-TOF/MS: m/z calcd for C48H54N12 [M+] 798.46; found
798.42. Anal. calcd For C48H54N12 (%): C, 72.15; H, 6.81; N,
21.04, found C, 72.07; H, 6.32; N, 21.61.
2(3),9(10),16(17),23(24)-Tetra(butylamino)phthalocyanine
(b-Pc). Yield, 25% (20 mg). UV-vis [lmax (nm) (log(3), Mꢂ1 cmꢂ1)]:
CHCl3, 340 (4.99), 447 (4.63), 667 (4.78), 741 (5.07). IR (KBr
pellet cmꢂ1): 3402(m), 3303(w), 2945(s), 2876(m), 2210(m),
1614(vs), 1505(s), 1346(m), 1276(w), 1117(m), 1008(m), 829(w),
760(m). 1H NMR (400 MHz, [D6]DMSO, 25 ꢀC, TMS, ppm): 8.95–
8.82 (4H, –PcC H –), 8.37–8.20 (4H, –PcC H –), 7.38–7.34 (4H,
6
3
6
3
–PcC H –), 6.96–6.79 (4H, –NH–), 3.44–3.30 ð ꢂ NHCH 2 ꢂ Þ,
6
3
1.81–0.93 (28H, –CH2CH2CH3). MALDI-TOF/MS: m/z calcd for
48H54N12 [M+] 798.46; found 798.75. Anal. calcd for C48H54N12
(%): C, 72.15; H, 6.81; N, 21.04, found C, 72.08; H, 6.42; N, 21.50.
C
Fig. 1 Left: Comparison in the electronic absorption spectra of a-Pc
(chloroform), b-Pc (chloroform), HSA-a-Pc NPs (water), and HSA-b-
Pc NPs (water), as well as HSA in aqueous solution (1.0 mg mLꢂ1). Right:
Fluorescence spectra of H2Pc(t-butyl)4 (0.5 mM, excited at 630 nm), a-
Pc (0.5 mM, excited at 680 nm), and b-Pc (0.5 mM, excited at 680 nm) in
chloroform as well as HSA-a-Pc NPs and HSA-b-Pc NPs (20 mM Pc
Synthesis of HSA-a-Pc and HSA-b-Pc NPs
2.0 mg of a-Pc was dispersed in 2.0 mL of dimethylacetamide
(DMAC). Then, 60 mL of the a-Pc/DMAC solution was mixed with equiv., excited at 680 nm) in aqueous solutions.
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inorganic oxide or organic molecules,27,28 was selected to their S1 uorescent emission at 812 and 761 nm were largely
prepare the HSA-involved a-Pc and b-Pc NPs. As is shown in quenched. They all possessed very low uorescence quantum
Scheme 1, aer separately combining the HSA aqueous solution yields (Fa-Pc ¼ 0.0045, Fb-Pc ¼ 0.0049) with respect to the
with the dimethylacetamide (DMAC) solutions of a-Pc and b-Pc, reference of tetrakis(tert-butyl)phthalocyanine H2Pc(t-butyl)4 (F
the HSA-Pc NPs of HSA-a-Pc and HSA-b-Pc were readily ¼ 0.77, chloroform), which is mainly due to the increased
produced (for details see the ESI†). The generated HSA-a-Pc and nonradiative decay rate associated with the p–p conjugation
HSA-b-Pc NPs were collected by centrifugation and washed with between the substituted N atoms and the central phthalocya-
water for three times before storing in water (Fig. S4†). The nine chromophore. The same phenomenon was previously
uploaded phthalocyanine amount in the obtained Pc NPs was observed in tetrakis(dibutylamino)phthalocyanine H2Pc
easily determined by the UV standard working curves aer [N(C4H9)2]4.24 Notably, aer formation of the HSA-Pc NPs, the S1
being extracted with organic solution (for details see the ESI, uorescence emission of HSA-a-Pc (F ¼ 0) and HSA-b-Pc (F ¼ 0)
Fig. S5 and S6†).
NPs were completely quenched due to the ACQ effect (Fig. 1).
As shown in Fig. 2, the transmission electron microscopy Some previous studies have shown that the S1 uorescence
(TEM) images of the HSA-a-Pc and HSA-b-Pc NPs indicated that emission quenching of Pcs would signicantly improve their
they all featured spherical particles with the diameter of ca. 60 light-to-heat conversion.8,11,12 Therefore, HSA-a-Pc and HSA-b-
and 30 nm respectively, which was further supported by the Pc herein are expected to be the potential photothermal mate-
dynamic light scattering (DLS) analysis (centered at 75 and rials for phototherapy of cancer under NIR laser irradiation.
35 nm respectively, Fig. S4†). The slight difference in size
should result from the solvation effect depending on the
different measurement.29 The suitable size of 35–75 nm with
negatively charged surface would enable the HSA-a-Pc and HSA-
b-Pc NPs to accumulate in the tumor sites through the
enhanced permeability and retention (EPR) effect.30 For bio-
logical application, the stability of HSA-a-Pc and HSA-b-Pc NPs
in ultrapure water, PBS (pH ¼ 6.5 and 7.4), and cell culture
media (RPMI 1640) was examined. As shown in Fig. S4,† they all
displayed excellent dispersity and stability in all media without
signicant macroscopic aggregates within ve days. Besides,
the measured z potentials of the HSA-a-Pc and HSA-b-Pc NPs
are ca. ꢂ15.4 and ꢂ17.8 mV respectively (Fig. S7†), which also
implied that these colloidal systems are stable and
biocompatible.
It is different from a-Pc and b-Pc monomers, the HSA-a-Pc
and HSA-b-Pc NPs in water displayed the broadened absorption
bands at 620–960 nm and 590–830 nm, respectively (Fig. 1).
Moreover, their maxima adsorptions were blue-shied with
respect to the a-Pc and b-Pc monomers in chloroform (Fig. 1),
suggesting that they might feature a face-to-face stacking mode
in the HSA-based NPs.
Photoproperties of HSA-a-Pc and HSA-b-Pc NPs
As shown above, the S1 uorescence emission of the HSA-a-Pc
and HSA-b-Pc NPs in water were signicantly suppressed, so
their excellent photothermal performance was expected.34
Fig. 3 showed that the photothermal behaviour of HSA-a-Pc
NP was concentration and light intensity dependent, and the
ꢀ
highest temperature increment (DT) of 34 C was observed at
a concentration of 80 mM (Pc equiv.) under 808 nm laser irra-
diation at 1.5 W cmꢂ2. In contrast, only ca. 3 C temperature
ꢀ
increase of pure water was detected in the absence of HSA-a-Pc
NP. This suggested that HSA-a-Pc was a highly efficient photo-
thermal conversion species which was able to convert light
energy into heat in an effective and quick way even irradiated by
laser at 808 nm. To determine the photothermal efficiency of
HSA-a-Pc, its heating and cooling curve in water was measured
(Fig. S8†). According to the obtained data, the light-to-heat
conversion efficiency h was calculated to be 56%, which is
higher than those of HSA-FePc (44%, 671 nm),35 DBCO-ZnPc-LP
(44%, 808 nm),36 O2@PFH@HMoSx-HSA/AlPc (40%, 670 nm),37
4OCSPC/F127 micelles (47%, 808 nm).38 Aer six cycles, the
photothermal heating capability for the HSA-a-Pc NP at 20 mM
It is known that phthalocyanine uorescence emission
included S1 (Q band, lowest state) and S2 (Soret, upper excited
state) emissions.31–33 As shown in Fig. 1, the uorescent emis-
sion spectra of both a-Pc and b-Pc in chloroform indicated that
(Pc equiv.) under 808 nm laser ꢀirradiation (1.5 W cmꢂ2
)
remained robust with DT up to 17 C, demonstrating its excel-
lent photostability. Similarly, the HSA-b-Pc NP also possessed
Fig. 3 Photothermal property of HSA-a-Pc NPs in aqueous solution
Fig. 2 TEM images of HSA-a-Pc NPs (left) and HSA-b-Pc NPs (right), under 808 nm laser irradiation. Left: Light intensity-dependent
scale bars: 50 nm. The insets present the picture of their Tyndall temperature increase at the concentration of 80 mM (Pc equiv.). Right:
phenomenon in aqueous solution, which further supports the TEM and Concentration-dependent temperature increase with a 0.9 W cmꢂ2
DLS analysis.
laser intensity.
25962 | RSC Adv., 2020, 10, 25958–25965
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an excellent photothermal conversion efficiency with h value of 69%) at the same concentration (20 mM, Pc equiv.). Their
ꢀ
54% and temperature increase of 17 C under the same condi- superior therapeutic effect was also directly demonstrated by
tions (808 nm, 1.5 W cmꢂ2, 20 mM Pc equiv.) (Fig. S9†). The high a live/dead cell co-staining study. As shown in Fig. 5c, only the
photothermal performance and high NIR photostability cells exposed to laser irradiation (within the cyan shadow) were
exhibited by HSA-a-Pc and HSA-b-Pc make them promising killed, and a clear demarcation line between live cells (green)
photothermal agents for phototherapy of cancer.
and dead cells (red) was observed in both cell lines (Fig. 5d).
In addition, the single oxygen generating ability of HSA-a-Pc Owing to the similar photothermal heating capability of HSA-a-
and HSA-b-Pc under NIR light (808 nm) illumination was Pc and HSA-b-Pc (20 mM, Pc equiv.) under 808 nm laser irradi-
investigated using DPBF as a detector by monitoring oxidation ation mentioned above, the higher phototoxicity exhibited by
of DPBF at 420 nm. Compared to HSA-b-Pc, HSA-a-Pc exhibited HSA-a-Pc was clearly attributed to the fact that HSA-a-Pc
more efficient singlet oxygen production (Fig. S10†), which generated more single oxygen and could kill cancer cells more
might be caused by the different location of peripheral group in efficiently via synergistic PDT/PTT under a single NIR laser
Pc.39 The singlet oxygen generation of the HSA-a-Pc and HSA-b- irradiation.
Pc NPs in cells upon 808 nm laser irradiation were also exam-
Recently, some very impressive works of Pc-nanomaterials
ined, and the result indicated that HSA-a-Pc also induced for PTT/PDT phototherapy have been reported, which are
higher singlet oxygen generation in MCF-7 cells than that of summarized in Table 1.9,10,13,37,40–42 Compared to the reported Pc-
HSA-b-Pc (Fig. 4).
based NPs, the phototherapy treatment of HSA-a-Pc in living
cells met a single NIR light source (808 nm), high light-to-heat
conversion efficiency h (56%) and tiny Pc amount (20 mM) for
less than 20% cell viability. So, HSA-a-Pc herein is in a strong
position among the Pc-based phototherapy nanomaterials.
To further investigate the phototherapy of HSA-a-Pc and HSA-b-
Pc NPs, the in vivo antitumor effects of Pc NPs were evaluated by an
MCF-7 xenogra model. Twenty-four nude mice bearing tumors
were randomly divided into six groups (Fig. 6 and S13†). For the
group of laser only, the tumor volume increased rapidly, and there
was no difference from the control group. Aer the intratumor
injection of HSA-a-Pc and HSA-b-Pc NPs (200 mM, Pc equiv., 50 mL)
In vitro and in vivo photothermal therapy
For examination of the cellular biocompatibility and membrane
permeability of HSA-a-Pc and HSA-b-Pc NPs, cell imaging
experiments were carried out against MCF-7 cells by confocal
laser scanning microscopy (CLSM). As is shown, both HSA-a-Pc
and HSA-b-Pc NPs displayed a weak S2 emission of Pc31–33 with
a maximum band at around 469 nm upon 405 nm irradiation
(Fig. S11†). Aer they (both at 20 mM, Pc equiv.) separately
incubated with MCF-7 cells for 2 h, MCF-7 cells were visualized
by the green emission from Pc (lex ¼ 405 nm). In addition, we
noticed that the green (440–480 nm) luminescence mainly
located in the cytoplasm, demonstrating that the HSA-a-Pc and
HSA-b-Pc NPs could readily pass across the tumour cell
membrane into the cytoplasm (Fig. S12†).
In addition, the in vitro phototoxicity of HSA-a-Pc and HSA-b-
Pc NPs were tested by using the standard 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assays
against MCF-7 cells at various concentrations (0–20 mM, Pc
equiv.) under irradiation at a power density of 1.5 W cmꢂ2 for
10 min. As shown in Fig. 5a, before laser irradiation, both HSA-
a-Pc and HSA-b-Pc NPs could maintain more than 90% cell
viability, indicating that they were biocompatible with negative
dark cytotoxicity. As shown in Fig. 5b, upon 808 nm laser irra-
diation (1.5 W cmꢂ2, 10 min), HSA-a-Pc showed a high photo-
toxicity of ca. 80% at the concentration of 20 mM (Pc equiv.) for
the tested cell line, which was higher than that of HSA-b-Pc (ca.
Fig. 5 In vitro cytotoxicities of HSA-a-Pc and HSA-b-Pc NPs against
MCF-7 cells before (a) and after (b) being irradiated with 808 nm laser
at a power density of 1.5 W cmꢂ2 for 10 min. (c) A sketch map of the cell
culture dish after incubation with Pc NPs. The cyan circle with shadow
shows the laser spot. (d) Confocal images of Calcein-AM (green, live
cells) and PI (red, dead cells) co-stained MCF-7 cells incubated without
Fig. 4 ROS generation induced by HSA-a-Pc and HSA-b-Pc (both at and with HSA-a-Pc and HSA-b-Pc NPs (both at 20 mM, Pc equiv.)
20 mM Pc equiv.) in MCF-7 cells in the presence of laser irradiation for before and after exposed to irradiation for 10 min (808 nm, 1.5 W
5 min (808 nm, 1.5 W cmꢂ2).
cmꢂ2).
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Table 1 Summary of recent typical Pc-nanomaterials for PTT/PDT synergistic phototherapy in living cells by a single light source
Sample
Light source (W cmꢂ2
)
h (%)
PS (mM)
MTT (%)
Ref.
Pc@HSNs
NanoPcTB
ZnPc NW
O2@PFH@HMoSx-HSA/AlPc
GR-TSCuPc
730 nm (1.5)
655 nm (2.5)
808 nm (3.0)
670 nm (1.0)
650 nm (3.0)
650 nm (0.7)
650 nm (3.0)
808 nm (1.5)
808 nm (1.5)
37
—
—
40
—
31
—
54
56
ꢃ575
6
ꢃ205
ꢃ255
ꢃ15
20
30
20
50
25
40
ꢃ15
15
31
20
9
10
13
37
40
41
42
ZnPc NPs
SWNHsꢂTSCuPc
ꢃ15
HSA-b-Pc
20
This work
This work
HSA-a-Pc
20
synergistic phototherapy upon a single NIR light source.
Moreover, the blood circulation and biodistribution of these
HSA-Pc NPs need to be examined in the future for clinical
applications.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
We are grateful for nancial support from NSFC (Grant No.
21501111, 21971153, and 21671122), the Taishan Scholar’s
Construction Project, and Natural Science Foundation of
Shandong Province (Grant No. ZR2014BP011 and ZR2016BQ25).
Fig. 6 Nude mice bearing MCF-7 tumors (n ¼ 24) were randomly
distributed into six groups when the tumor size reached ꢃ140 mm3.
Light treatment was performed on the tumor site using an 808 nm
laser (1.5 W cmꢂ2, 8 min) for therapy. (a) Photographs of tumor tissue
obtained after treatment. (b) Tumor volume of the nude mice in
various groups during the treatment. (c) Tumor weight obtained after
treatment. All data are presented as the mean ꢄ SD (n ¼ 4).
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