Photoswitchable phthalocyanine-assembled nanoparticles for controlled "double-lock" photodynamic therapy

Article abstract of DOI:10.1039/c9cc03960k

In the current study, a new nanoparticle platform, NanoAzoPcS, is created by co-assembly of phthalocyanine and azobenzene amphiphiles, which can be used to gain precise control of PDT simply by regulating the stoichiometric ratio of the components and usi

Full text of DOI:10.1039/c9cc03960k

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Photoswitchable phthalocyanine-assembled
nanoparticles for controlled ‘‘double-lock’’
photodynamic therapy†‡
Cite this: Chem. Commun., 2019,
55, 12316
Received 12th August 2019,
Accepted 17th September 2019
Hong-Bo Cheng,§a Xingshu Li,§a Nahyun Kwon,§a Yanyan Fang,*b Gain Baeka and
a
Juyoung Yoon
*
DOI: 10.1039/c9cc03960k
rsc.li/chemcomm
In the current study, a new nanoparticle platform, NanoAzoPcS, an important role in regulating the physical, chemical and/or
is created by co-assembly of phthalocyanine and azobenzene biological properties of fluorescent dyes.7 Moreover, control of
amphiphiles, which can be used to gain precise control of PDT the packing of functional dyes in the supermolecular aggregates
simply by regulating the stoichiometric ratio of the components serves as a facile method to regulate and control photophysical
and using light irradiation. The results of antibacterial studies show properties. This feature enables the formation of new optical tools
that NanoAzoPcS serves as a smart PS for controlled PDT.
from individual dye molecules.8
Zinc(II) phthalocyanines (ZnPcs) have unique advantages for
Owing to its high spatiotemporal selectivity, photodynamic use in phototherapies, especially in medical applications of
therapy (PDT) has become a clinically promising approach for PDT, because they have long absorption wavelength maxima
the treatment of noninvasive cancers and microbial infections.1 and high molar extinction coefficients.9 However, the fact that
Upon light irradiation, preferably in the phototherapeutic Znpcs always exists in ‘‘on’’ states severely limits their use in
window (650–850 nm), certain dyes (photosensitizers, PSs) are PDT. In addition, control of the supramolecular assembly of
transformed to their triplet excited states through intersystem Znpcs is complex and uncertain. Precise regulation of the
crossing (ISC) and then generate highly cytotoxic, short-lived construction of assemblies of this type is critical to promote
reactive oxygen species (ROS).2 The ROS and products of their an efficient therapeutic effect and to have low side effects.
chemical reactions only form at the time of irradiation and only
Moreover, because they have highly-conjugated organic
in the region that is exposed to light. As a result, PDT possesses structures, ZnPcs display poor water solubilities, which detracts
unique advantages associated with spatiotemporal selectivity, from their use in clinical medicine.10 However, introducing
which benefit clinical applications.3 However, PDT suffers from hydrophilic groups into ZnPcs enhances their water solubilities.
several problems including the low water solubility of current In earlier studies, we showed that amphiphilic analogues of
molecular PSs (such as porphyrin and phthalocyanine), low phthalocyanines can be used in theranostic approaches owing to
photodynamic efficiency of nanostructured PSs and lack of efficient their high photodynamic activities toward cancer cells.11
methods for delivery of PSs to target cells and tissues.4
The concept of photopharmacology has been explored in the
Recently, supramolecular self-assemblies have emerged as context of various therapeutic applications.12 Azobenzenes,
promising tools in nanomedicine because they can serve as which have been used in photopharmacology,13 undergo well-
therapeutic nanosystems that can be controlled at the molecular known reversible NQN bond E–Z isomerization reactions that can
level.5 Self-assembly of functional dyes is a versatile, ‘‘bottom-up’’ be employed to control p–p stacking interactions.14 Because supra-
method for construction of nanodrugs for noninvasive cancer molecular coassembled nanoarchitectures have attracted increasing
therapy and microbial infections.4b,6 Molecular aggregation plays attention in the field of biomedicine, substances like azobenzenes
that undergo p–p stacking have been screened to identify guests
for forming coassembled nanoarchitectures with ZnPcs. How-
ever, to the best of our knowledge, activatable supramolecular
coassembled azobenzenes/ZnPcs-based PDT systems that enable
controlled ROS generation have not been developed to date.
In the study described below, we designed and synthesized
several novel amphiphilic azobenzenes (Azos), and explored their
ability to regulate the self-assembly behavior of 4-sulfonatophenoxy-
substituted ZnPcS (Fig. 1). Moreover, we showed that the new
a Department of Chemistry and Nano Science Ewha Womans University,
Seoul 120-750, Korea. E-mail: jyoon@ewha.ac.kr
b Beijing National Laboratory for Molecular Sciences, Key Laboratory of
Photochemistry, Institute of Chemistry, Chinese Academy of Sciences,
Beijing 100190, China. E-mail: fyy001@iccas.ac.cn
† This paper is dedicated to Prof. Eric V. Anslyn on the occassion of his 60th
birthday.
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc03960k
§ These authors contributed equally to this work.
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Fig. 1 Schematic illustration of the preparation of self-assembled Azo–
Znpcs nanoparticles (NanoAzoPcS) and their responsive disassembly
response to light for promotion of their photodynamic antibacterial
effects.
nanoparticles, NanoAzoPcS, formed from ZnPcS and Azos can be
used as light controlled antibactericidal agents.
The routes employed for the synthesis of ZnPcS and Azos 1–3 are
described in the ESI.‡ Several methods were used to examine
Fig. 2 (a) Models for assemblies of host ZnPcS and guest azobenzenes.
the formation of host–guest nanosystems by mixing ZnPcS and Azos Geometries were optimized by using the molecular mechanics and a
dreiding force field. Simulations were performed using Material Studio
1–3. Based on the results of calculations, which predict the strengths
of p–p stacking interactions and molecular distances (Fig. 2a), Azos 1
was expected to be an ideal guest for formation of coassembled
software packages from Accelrys Inc. (b) Changes in the absorption
spectrum of ZnPcS (5 mM) in water in the presence of (b) 0–10 and
(c) 10–35 mM Azo 1 (from). Changes in fluorescence spectrum of host ZnPcS
host–guest nanosystems with ZnPcS. The binding affinities between
ZnPcS and Azos 1–3 were determined by using UV-vis titration.
Inspection of Fig. 2b shows that addition of 2 equiv. of Azo 1 to
ZnPcS causes the greatest change in the absorption spectrum of
(5 mM) in water in the presence of (d) 0–10 and (e) 10–35 mM of Azo 1.
Dynamic light scattering (DLS) analysis was used to monitor
ZnPcS. Interestingly, after addition of more than 2 equiv. of Azo 1, the formation of the coassembled host–guest nanosystems
the intensity of the absorption of ZnPcS in the range of 600–800 nm ZnPcS–azo 1. The results (Fig. 3a) show that pure ZnPcS and
undergoes decreases (Fig. 2c), which based on Kasha’s exciton theory NanoAzoPcS (ZnPcs/azo 1 = 5 mM/5 mM) in water have hydro-
indicates that a decrease and broadening of the Q-band occurs. dynamic diameters that are less than 20 nm. However, the
However, Azos 2–3 induces only slight changes in the absorption assembly formed by mixing ZnPcS and 7 equiv. of Azo 1 has a
spectrum of ZnPcS owing to unfavorable stacking and molecular large mean hydrodynamic diameter of ca. 120 nm (Fig. 3b).
distances, which decrease the propensity for complex formation Moreover, NanoAzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) in phos-
(Fig. S5 and S6, ESI‡).
phate buffered solution (PBS) has a size distribution that is
Fluorescence changes caused by mixing host ZnPcS and similar to that in water.
guest Azos 1–3 were explored in order to obtain information about
Transmission electron microscopy (TEM) was utilized to
aggregate generation. As shown in Fig. 2d and e, H-aggregates obtain more information about the formation of NanoAzoPcS.
exist in the coassembled host–guest nanosystem ZnPcS–Azo 1. In As can be seen by viewing the TEM images in Fig. 3c, Nano-
addition, 2 equiv. of Azo 1 induces the largest enhancement of AzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) has an oval-shaped struc-
ZnPcS fluorescence. Moreover, unlike ZnPcS, NanoAzoPcS ture with a size of ca. 110 nm, which is in accordance with the
(ZnPcs/azo 1 = 5 mM/35 mM) does not fluoresce. This finding size determined using DLS.
demonstrates that strong interactions occur between ZnPcS and
Stability and size distribution are key factors determining
Azo 1 in the assembly. In striking contrast, Azo 2 and Azo 3 do the practical utility of biomaterials. Hence, the stability of
not induce enhancements in the fluorescence of ZnPcS (Fig. S7 NanoAzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) in water and PBS
and S8, ESI‡). Clearly, the combined results demonstrate that was assessed. As can be seen by viewing the images in Fig. 3d,
Azo 1 interacts with ZnPcS most strongly to form a host–guest NanoAzoPcS is stable in both water and PBS. The high stability
nanosystem.
of NanoAzoPcS in PBS (physiological conditions) indicates its
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Fig. 3 Self-assembly to form NanoAzoPcS and its stability. (a) DLS size
profiles for ZnPcS (5 mM) and NanoAzoPcS (ZnPcs/azo 1 = 5 mM/5 mM).
(b) Size distribution of NanoAzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) in water
and PBS determined by using DLS. (c) The morphology of NanoAzoPcS
(ZnPcs/azo 1 = 5 mM/35 mM) determined by using TEM. (d) Mean size of
NanoAzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) in water and in PBS after aging
for different times detected by using DLS. (e) Zeta potential of NanoAzoPcS
(ZnPcs/azo 1 = 5 mM/35 mM) in water and PBS.
Fig. 4 Photoswitchable self-assembly process of NanoAzoPcS for con-
trolled photoactivity. (a) Absorption and (b) fluorescence spectra (excited
at 610 nm) of NanoAzoPcS (ZnPcs/azo 1 = 5 mM/5 mM) after photoirradia-
tion at 365 nm. (c) Possible structures of the assembly of NanoAzoPcS
after photoirradiation at 365 nm and then at 450 nm. (d) Changes in the
fluorescence intensity of NanoAzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) in
water upon alternating UV (365 nm) and visible light (450 nm) irradiation.
(e) ROS generation by (365 nm) UV irradiation of NanoAzoPcS (ZnPcs/azo
1 = 5 mM/35 mM) in water.
potential for use in biomedical applications. The data in Fig. 3e
show that the zeta potential of NanoAzoPcS is ca. +27 mV in
water and ca. +23 mV in PBS, which indicates its high disper- and Fig. S11, ESI‡). It is known that singlet excited states of PSs
sion stability. undergo decay through three pathways including fluorescence
Azobenzene containing host–guest nanosystems have super- emission, generation of heat through vibrational relaxation and
ior features for applications as supramolecular biomaterials. production of the corresponding ROS forming triplet excited
An important reason for this is that noncovalent interactions states by intersystem crossing (ISC). 2,7-Dichlorofluorescin dia-
between azobenzenes and other substances can be controlled cetate was used as a probe in order to explore NanoAzoPcS
by using light.15 Therefore, disassembly of azobenzene based (ZnPcs/azo 1 = 5 mM/35 mM) promoted generation of ROS in
supramolecular assemblies has the capability of being triggered water. Importantly, the results show that 365 nm irradiation of
by an external light stimulus that promotes E–Z isomerization.16 NanoAzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) leads to a significant
The photophysical and photochemical properties of NanoAzoPcS increase in the formation of ROS such as 1O2 (Fig. 4e and Fig. S12,
were evaluated in order determine if it can be employed as a light S13, ESI‡).
controlled PS for PDT. Analysis of the spectra shown in Fig. S9
To assess the ability of NanoAzoPcS to serve as a novel
and S10 (ESI‡) indicates that an isosbestic point exists at 442 nm nanoPS for PDT, we investigated its use in antibacterial photo-
and that a significant decrease in the intensity of the absorption dynamic therapy (APDT). The Gram-negative bacteria E. coli
maximum at 358 nm occurs upon 365 nm irradiation. This and Gram-positive bacteria S. aureus in a nutrient medium were
observation shows that photoinduced E–Z isomerization occurs used as model biological targets. The results demonstrate that
in the azo 1 component. In addition, the absorption maximum NanoAzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) is well dispersed in
of NanoAzoPcS at 355 nm undergoes a significant decrease while water and PBS before and after irradiation with 365 nm light.
at the same time the intensity of the original absorption band in A PBS solution of NanoAzoPcS was added to the E. coli and
the 600–800 nm range is recovered.
S. aureus cells and the cells were then incubated for 2 h. The
Moreover, the intensity of fluorescence of NanoAzoPcS Cryo TEM images of S. aureus bacterial cells show that small
dramatically increases upon irradiation at 365 nm (Fig. 4d), nanoparticles after dissociation of NanoAzoPcS (ZnPcs/azo 1 =
which leads to disruption of p–p stacking between ZnPcS and 5 mM/35 mM) adhere to the surface of the bacterial cells
azo 1 (Fig. 4c). After three cycles of irradiation with UV (365 nm, (Fig. S14, ESI‡). Following irradiation of the treated bacterial
for 1.0 min) and visible light (450 nm for 5.0 min), photo- cells using a 655 nm laser (655 nm, 0.4 W cmÀ2, 10 min), the
switchable NanoAzoPcS maintains a high performance (Fig. 4d cells were plated on LB agar plates and the plates were incubated
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Conflicts of interest
There are no conflicts to declare.
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Fig. 5 Controlled photodynamic antibacterial effects. Photographs of
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Azo 1 and NanoAzoPcS do not promote inhibition of bacterial
growth both in the absence and presence of 655 nm laser irradiation.
However, unlocking NanoAzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) does
have inhibitory effects on the growth of 655 nm laser irradiated
E. coli and S. aureus cells (Fig. 5 and Fig. S15, ESI‡). Moreover, as can
be seen by viewing Fig. S16 (ESI‡), the antibacterial activity of
unlocked NanoAzoPcS (ZnPcs/azo 1 = 5 mM/35 mM) is concentration
dependent in the tested range of 100–1000 nM. The number of
colony forming units (CFUs) was used to determine the numbers of
live S. aureus bacterial cells. Inspection of Fig. S16 (ESI‡) shows that
‘‘double-lock’’ NanoAzoPcS has antibacterial effects on S. aureus in
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J. Y. is thankful for financial support from the National
Research Foundation of Korea (NRF), which is funded by the
Korea government (MSIP) (No. 2012R1A3A2048814).
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