A 2-pyridone modified zinc phthalocyanine with three-in-one multiple functions for photodynamic therapy

Article abstract of DOI:10.1039/d1cc00645b

A 2-pyridone modified zinc phthalocyanine (denoted ZnPc-PYR) achieves a one stone for three birds outcome in the photodynamic therapy (PDT) treatment of cancer. ZnPc-PYR can be excited by both 665 and 808 nm light to treat superficial and deep tumors, store and slowly release singlet oxygen (¹O₂) to improve its utilization and downregulate the HIF-1 (hypoxia-inducible factor 1) expression level to enhance the tumor cell's sensitivity to PDT treatment under hypoxic conditions.

Full text of DOI:10.1039/d1cc00645b

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A 2-pyridone modified zinc phthalocyanine with
three-in-one multiple functions for photodynamic
therapy†
Cite this: Chem. Commun., 2021,
57, 3127
Received 3rd February 2021,
Accepted 12th February 2021
ab
Yanqing Li,‡a Chongchong Wang,‡a Lin Zhou *a and Shaohua Wei
*
DOI: 10.1039/d1cc00645b
rsc.li/chemcomm
A 2-pyridone modified zinc phthalocyanine (denoted ZnPc-PYR) lead to an insufficient effect of PDT in treating deep lesions. In
achieves a one stone for three birds outcome in the photodynamic contrast, the penetration depth and transfer efficiency of near-
therapy (PDT) treatment of cancer. ZnPc-PYR can be excited by infrared (NIR) light are efficient for deep-site lesion treatment.
both 665 and 808 nm light to treat superficial and deep tumors, Therefore, PSs that can be excited by both visible and NIR light
store and slowly release singlet oxygen (1O2) to improve its utiliza- could effectively treat superficial and deep tumors.
tion and downregulate the HIF-1 (hypoxia-inducible factor 1)
1O2 is the primary ROS for most of the PSs in PDT. However,
expression level to enhance the tumor cell’s sensitivity to PDT its lifespan is incredibly short (o40 ms).7,9,10 Thus, many O2
1
treatment under hypoxic conditions.
are quenched before oxidative damage to tumor cells. There-
fore, the inadequate utilization of 1O2 would significantly
The mechanical research and clinical application of PDT for reduce the therapeutic effect of PDT.
cancer treatment have been tremendously developed in recent
Besides, hypoxia is a unique characteristic of the tumor
years. The PDT mechanism refers to a process in which a microenvironment. The PDT process consumes O2 to aggravate
photosensitizer (PS) accumulated in the tumor tissue is excited the tumor hypoxia further and eventually lead to a poor PDT
by laser irradiation of a specific wavelength. The excited photo- outcome. Moreover, under hypoxic conditions, HIF-1 is up-
sensitizer transfers energy to the surrounding O2 to generate regulated. HIF-1 upregulation is a predominant event in the promo-
highly toxic reactive oxygen species (ROS) to damage the tumor tion of cancer progression, such as angiogenesis, metastasis, and
tissue. Light, PS, and O2 are the three critical elements in apoptosis resistance.11,12 HIF-1 up-regulation could enhance the
PDT.1,2 The ROS generation capacity and utilization efficiency cells’ resistance to various cancer treatments, including PDT. The
are essential indicators to evaluate the PDT activity. It has been HIF-1 expression level is positively correlated with patient mortality
found that the insufficient penetration depth of the light,3,4 the and treatment tolerance. Thus, HIF-1 inhibition can enhance the
tumor hypoxic microenvironment,5,6 and the short lifespan of cancer cell’s sensitivity to PDT to enhance the treatment efficiency
1
some ROS, particularly, O2,7 restrict the PDT performance.
under hypoxic conditions.13,14
The excitation wavelength of classic PSs is in the range of
ZnPc is a representative of a third-generation PS. Based on
visible light, for which the tissue penetration depth is insuffi- the above three bottlenecks in PDT treatment, we designed and
cient. Also,8 hemoglobin and melanin in tissues have strong synthesized a 2-pyridone modified ZnPc (denoted ZnPc-PYR)
absorption of light from 600 to 700 nm, inducing a reduction in with multiple functions (Fig. 1 and Fig. S1, S3, ESI†).
the transfer efficiency of light. All of the above problems will
First, the ZnPc-PYR can be excited by both 665 and 808 nm
light to generate ROS. Compared with the 665 nm light, the
808 nm light has a deep penetration depth in tumor tissue.
Therefore, the ZnPc-PYR could achieve high-efficiency PDT treat-
ment of superficial and deep tumors, simultaneously. Second, the
ZnPc-PYR was modified by four 2-pyridones. 1O2 can oxidize
a College of Chemistry and Materials Science, Jiangsu Key Laboratory of
Biofunctional Materials, Jiangsu Collaborative Innovation Centre of Biomedical
Functional Materials, Key Laboratory of Applied Photochemistry,
Nanjing Normal University, Nanjing, Jiangsu 210023, China.
E-mail: zhoulin@njnu.edu.cn, shwei@njnu.edu.cn
1
2-pyridone to its internal peroxide to store O2. Then, the stored
b School of Chemistry and Chemical Engineering, Yancheng Institute of Technology,
1O2 was gradually released under 37 1C to improve the utilization
Yancheng, Jiangsu 224051, China
1
of O2. Third, the ZnPc-PYR can significantly down-regulate the
† Electronic supplementary information (ESI) available: Materials and methods,
the total ROS and 1O2 generation of ICG, some in vitro toxicity data and in vivo
biocompatibility. See DOI: 10.1039/d1cc00645b
HIF-1 level to enhance the tumor cell’s sensitivity to PDT treat-
ment under hypoxic conditions.15–17 Our research indicated that
ZnPc-PYR exhibited a satisfactory PDT outcome for cancer
‡ Y. Q. Li and C. C. Wang contributed equally to the work.
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Fig. 1 Schematic diagram of ZnPc-PYR with three-in-one multiple func-
tions for tumor PDT.
Fig. 2 (A) Absorbance spectra of ZnPc-PYR and the schematic presenta-
tion of its potential application in the superficial and deep tumor PDT
treatment. (B) The 1O2 (1) and total ROS (2) generation in aqueous solution
of ZnPc-PYR post 665 nm light irradiation. (C) The 1O2 (1) and total ROS (2)
generation in aqueous solution of ZnPc-PYR post 808 nm light irradiation.
(D) In vitro ROS generation of ZnPc-PYR under various light irradiation
conditions (bar = 20 mm).
treatment. The above positive results provided a reference for
research on the design and synthesis of multiple function inte-
grated PSs against the bottleneck of PDT treatment.
The water solubility of ZnPc-PYR is low. Thus, the stock
solution of ZnPc-PYR was prepared in DMSO. After addition to
an aqueous solution, ZnPc-PYR tends to aggregate leading to
precipitation. CrEL (Cremophor EL) is a non-ionic surfactant
with excellent solubility and emulsifying activity. CrEL is widely and Drug Administration (FDA) for NIR-II (1000–1700 nm)
used to improve drug solubility since it is a nontoxic and diagnosis using its tail fluorescence in the NIR-II region.24
nonirritating substance in various acute toxicity and chronic
We proved that ZnPc-PYR in the presence of CrEL is taken
toxicity tests.18,19 Thus, CrEL was used as a solubilizer of ZnPc-PYR up by cells through endocytosis mediated by caveolae and
to improve its water dispersion ability and activity (Scheme S1 and micropinocytosis (Fig. S7, ESI†). The in vitro ROS generation
Fig. S4, S5, ESI†). And in all of the chemical and activity studies for ability of ZnPc-PYR was studied using DCFH-DA as a probe.
ZnPc-PYR, 0.001% CrEL was added.
Similarly, ZnPc-PYR can generate ROS effectively inside cancer
The classic ZnPcs are activated by red light, which has a cells post irradiation by 665 and 808 nm light. Besides, the total
limited tissue-penetration depth and is suitable to treat super- ROS level in 665 + 808 nm light irradiation cells was signifi-
ficial cancer. In contrast, NIR (700–1000 nm) has greater cantly higher than that of 665 or 808 nm alone treated cells
penetration depths than red light and is suitable to treat large (Fig. 2D). Notably, 808 nm light can penetrate 1.5 cm pork to
and deep-seated tumors.20,21
trigger ZnPc-PYR to generate ROS inside cancer cells. There-
As shown in Fig. 2A, ZnPc-PYR has strong absorbance fore, ZnPc-PYR can be used in a dual-wavelength treatment for
around 665 nm, indicating that it can be excited by red light superficial and deep tumors.
to generate ROS for superficial cancer treatment. Interestingly,
The above results showed that the ZnPc-PYR could generate
1
1
ZnPc-PYR also has tail absorbance around 800 nm. We adequate O2 under 665 or 808 nm light irradiation. O2 is the
proposed that it could be excited by NIR light to generate primary cytotoxic ROS in PDT. However, its lifetime is very
ROS for deep tumor treatment. To verify this hypothesis, we short (o40 ns).7 Therefore, many 1O2 would be quenched
detected the ROS generation ability of ZnPc-PYR under 665 and before damaging the target biomolecules, resulting in low
1
808 nm light irradiation using ADPA (a disodium salt of 9, utilization of O2, limiting the PDT efficiency. 2-Pyridone can
1
10-anthracenedipropionic acid) and DCFH (dichlorofluorescin be oxidized by O2 leading to endoperoxide formation, which
1
diacetate) as a O2 and total ROS probe, respectively.
could gradually release 1O2 under 37 1C. Thus, ZnPc-PYR
As shown in Fig. 2B and C, obviously 1O2 and total ROS should have 1O2 storage and gradual release functions to make
generation were detected by ZnPc-PYR post 665 and 808 nm full use of O2 (Fig. S8A, ESI†).25,26 Moreover, in order to verify
1
light irradiation. The ZnPc-PYR absorbance intensity at 808 nm that the 2-pyridone modifier is critical to the storage and
1
is not strong enough. So, we worry that its NIR light-triggered release of O2, we synthesized a phenyl-modified ZnPc (ZnPc-
PDT activity may not be strong enough. To solve this concern, BZA, Fig. S2, ESI†) as a negative control.
we compared the 1O2 and total ROS generation ability between
The 1O2 storage and release function of ZnPc-PYR was
ZnPc-PYR and ICG (Indocyanine Green), a classic NIR-I light- studied by monitoring the oxidation-caused absorption
triggered PS and photothermal agent, under 808 nm light decrease of ADPA in aqueous solution. As shown in Fig. S9
irradiation.22,23 As shown in Fig. S6 (ESI†), the results indicated and S10 (ESI†), for ZnPc-BZA, the absorption peak of ADPA at
that these abilities of ZnPc-PYR were higher than those of ICG. 378 nm (lmax of ADPA) decrease can only be detected under the
Tail absorbance2 or fluorescence is widely used for cancer light irradiation cycle. In the dark incubation cycle (37 1C) of
treatment or diagnosis. For example, the NIR-I (700–1000 nm) ZnPc-BZA, the absorption intensity decrease was not observed,
fluorescent ICG has been approved by the United States Food but an increase in the absorption value was observed. ADPA
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can be oxidized by O2 to its endoperoxide formation to store
1O2 and induce its absorbance to decrease. The 1O2 release
from endoperoxide formation to revert to ADPA induced the
above absorption enhancement phenomenon. However, the
released 1O2 from the endoperoxide formation is not enough
to oxidize ADPA. In contrast, for ZnPc-PYR, the decrease of the
absorption peak of ADPA at 378 nm can be detected under a
light irradiation cycle (665 nm light irradiation) and dark
incubation cycle. These results indicated that the ZnPc-PYR
possesses an efficient 1O2 storage and release function, and the
2-pyridone modification is critical for this function.
The in vitro 1O2 generation detection further proved the
1O2 storage and release function of ZnPc-PYR, both under 665
1
and 808 nm light irradiation. The in vitro O2 generation was
detected using SOSG as a probe using CLSM and FCM technology.
As shown in Fig. S8B (ESI†), a large amount of 1O2 was generated
immediately in cancer cells by ZnPc-PYR after irradiation by 665 or
808 nm light. After all the cells had been continuously incubated in
the incubator (37 1C) for 6 hours in the dark, the cells continued to
show enhancement of the fluorescence intensity, indicating that
1O2 can be continuously released in these cells.
Hypoxia is a primary characteristic of the solid tumor
microenvironment. The PDT process consumes O2 to aggravate
tumor hypoxia.27 HIF-1 is composed of a constitutively expressed
subunit (HIF-1b) and an O2-regulated subunit (HIF-1a). The up-
regulation of HIF-1 is a predominant event in cancer progression
promotion, such as angiogenesis, metastasis, and apoptosis
resistance. More significantly, HIF-1 upregulation is associated
with PDT treatment resistance. Thus, HIF-1 inhibition is an
effective way to enhance the PDT treatment efficiency.13,14
As shown in Fig. 3A, after treating with ZnPc-PYR, the
Fig. 3 (A) The expression (1) and quantitative comparison (2) of HIF-1a by
the western blot method post various treatments. The quantitative analysis
of apoptotic cells in various stages (1), and cancer cell killing capability (2),
of ZnPc-PYR post various kinds of light irradiation treatments under
normoxic (B) and hypoxic (C) conditions in HeLa cells. (Data are expressed
as means Æ SD; *P o 0.05; **P o 0.01 ***P o 0.001 versus control;
#P o 0.05; ##P o 0.01; ###P o 0.001 versus ZnPc-PYR + 665 + 808 nm
group.)
expression level of HIF-1a in cancer cells was significantly MTT assay. The PDT induced cell apoptosis was studied using a
down-regulated. In contrast, DMSO or CrEL did not influence PI/Annexin IV-FITC kit by FCM. The dark cytotoxicity test of
HIF-1a expression. The above positive results implied that ZnPc-PYR, CrEL and ICG indicated that they do not have dark
ZnPc-PYR could enhance cancer cell sensitivity to PDT treat- toxicity under our experiment conditions (Fig. S13, ESI†). As
ment and possess satisfactory PDT activity under hypoxic shown in Fig. 3B and Fig. S14A, S15 (ESI†), under normoxic
conditions.
conditions, ZnPc-PYR has 665 and 808 nm light triggering PDT
ICG has a maximum absorption band of around 800 nm. In activity to induce significant cancer cell apoptosis and cell
contrast, just as mentioned above, the ZnPc-PYR absorbance death. Moreover, the in vitro PDT activity of the dual-light
intensity at 808 nm is not strong enough. To verify that the NIR treatment (665 nm + 808 nm) group was higher than that of
light-triggered PDT activity of ZnPc-PYR is sufficient, we com- the single-light treatment groups. The in vitro photodynamic
pared the 808 nm light-triggered cancer cell killing ability activities of ZnPc-PYR were also examined in the presence of
between ICG and ZnPc-PYR. As shown in Fig. S11 (ESI†), post 1.5 cm pork to prove its deep tumor treatment potential
808 nm light irradiation, although the cancer cell killing ability (Fig. S16, ESI†).
of ZnPc-PYR is lower than that of ICG, about 50% of cancer cells
Moreover, just as mentioned above, the HIF-1 downregula-
were killed in the ZnPc-PYR treated group. Besides ROS, ICG tion function of ZnPc-PYR could enhance the cancer cell
could effectively absorb NIR light and convert it into heat to sensitivity to PDT treatment under hypoxic conditions. The
achieve photothermal therapy (PTT).28 ZnPc-PYR did not have a results in Fig. 3C and Fig. S14B (ESI†) verified this hypothesis.
photothermal conversion capability. Besides, pretreating by Under the hypoxic conditions, ZnPc-PYR also has satisfactory
ascorbic acid, an antioxidant regent, can inhibit the cancer cell dual-light induced PDT activity in HeLa cells. The same experi-
killing activity of ZnPc-PYR post 808 nm light irradiation. Thus, ment was also implemented on 4T1 cells, and similar results
the cancer cell killing mechanism by ZnPc-PYR post 808 nm were obtained (Fig. S17, ESI†). This property is significant for
light irradiation is a PDT process but not a PDT-PTT combi- its in vivo PDT treatment since the hypoxic and normoxic areas
nation process (Fig. S12, ESI†).
coexist in the solid tumor.
We evaluated the dual-wavelength triggered in vitro PDT
Based on the above positive in vitro results, we proposed that
effect. The in vitro cancer cell killing capability was studied by ZnPc-PYR would have satisfactory tumor suppression capability
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significantly damages tumor cells even under 1% O2 hypoxic
conditions. Therefore, ZnPc-PYR can achieve efficient treatment
of tumors based on the above multiple functions.
Natural Science Foundation of China (grant number
21671105), the project (grant number BK20161554) supported
by NSF of Jiangsu Province of China, the Priority Academic
Program Development of Jiangsu Higher Education Institu-
tions, and the Foundation of Jiangsu Collaborative Innovation
Centre of Biomedical Functional Materials (grant number
161090H001) are acknowledged.
Fig. 4 (A) Tumor volume changes post 14 days of various treatment. (B) The
final tumor volume comparison of various groups. (Data are expressed as
means Æ SD; *P o 0.05; **P o 0.01 ***P o 0.001 versus control; #P o 0.05;
##P o 0.01 ###P o 0.001 versus ZnPc-PYR + 665 + 808 nm group.)
Conflicts of interest
There are no conflicts to declare.
in vivo. As shown in Fig. S18 (ESI†), no noticeable weight loss or
main organ damage (from the H&E staining results of the tissue
sections) were detected during 14 days of ZnPc-PYR treatment
under various light irradiation conditions, indicating its ideal
biocompatibility. During the 14 days of treatment, the tumor
volumes of various groups were recorded and compared. As
shown in Fig. 4A and B, 665 and 808 nm light irradiation at the
tumor tissue did not induce tumor suppression, indicating that
the light dose used in this experiment is safe.
In contrast, both 665 and 808 nm light irradiation at the
tumor tissue can suppress tumor volume growth. Moreover, the
combination treatment of the dual-light group has the best
tumor suppression efficiency. Besides, the lesion damage
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