Doxorubicin-Loaded Photosensitizer-Core pH-Responsive Copolymer Nanocarriers for Combining Photodynamic Therapy and Chemotherapy

Article abstract of DOI:10.1021/acsbiomaterials.6b00762

Photodynamic therapy (PDT) is an emerging method for the treatment of cancer. Combination of PDT and chemotherapy is a hot topic though the two therapies could not simultaneously exert their perfect effect in vivo. Here we report a doxorubicin-loaded photosensitizer-core pH-responsive copolymer nanocarrier with high tumor targeting and anticancer effects due to integration of PDT with chemotherapy. The pH-responsive photosensitizer-core four-Armed star-shaped copolymer, [methoxy-poly(ethylene glycol)-poly(2-(N,N-diethylamino)ethyl methacrylate)-poly(?-caprolactone)]₄-zinc β-Tetra-(4-carboxyl benzyloxyl)phthalocyanine (PDCZP), was prepared, which was a molecular spherical nanocarrier in aqueous media. The carriers changed from small at high pH to large at low pH (51, 105, and 342 nm at pH 7.4, 6.5, and 5.0, respectively) and the zeta potential gradually increased (7.15, 16.2, and 26.1 mV at the above pH, respectively). PDCZP had a longer emission wavelength (max. 677 nm) than the parent photosensitizer, favoring light penetration through biological tissues. The singlet oxygen (¹O₂) quantum yield of PDCZP was 0.41. Doxorubicin (DOX) showed rapid release from PDCZP in the acidic media. More importantly, the drug-loaded nanocarriers showed the better in vitro and in vivo anticancer effects under lighting on MCF-7, SW480 cells and HepG2 cells and the murine hepatocellular carcinoma H₂₂ models than the other groups. PDCZP showed a high tumor targeting effect based on the enhanced permeation and retention effect and its small size. The photosensitizer-core nanocarrier is a promising photodynamic nanocarrier for integrating other therapies.

Full text of DOI:10.1021/acsbiomaterials.6b00762

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Article
A Doxorubicin-Loaded Photosensitizer-Core pH-Responsive Copolymer
Nanocarrier for Combining Photodynamic Therapy and Chemotherapy
Xiaohan Zhang, Qiu Li, Xiaodong Sun, Baolei Zhang, Hongxiang Kang, Fuli Zhang, and Yiguang Jin
ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00762 • Publication Date (Web): 15 Feb 2017
Downloaded from http://pubs.acs.org on February 19, 2017
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A DoxorubicinꢀLoaded PhotosensitizerꢀCore
pHꢀResponsive Copolymer Nanocarrier for
Combining Photodynamic Therapy and
Chemotherapy
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Xiaohan Zhang,†,‡ Qiu Li,†,¶ Xiaodong Sun,†,‡ Baolei Zhang,†,‡ Hongxiang Kang,† Fuli
Zhang,‡ and Yiguang Jin*,†,‡
†Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, 27
Taiping Road, Beijing 100850, China, eꢀmail address: jinyg@sina.com
‡Institute of Pharmacy, Pharmaceutical College of Henan University, Kaifeng 475004,
China
¶State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
Medical Sciences, University of Macau, Avenida Padre Tomas Pereira, Taipa, Macao
SAR, China
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ABSTRACT: Photodynamic therapy (PDT) is an emerging method for the treatment
of cancer. The combination of PDT and chemotherapy is a hot topic though the two
therapies could not simultaneously exert their perfect effect in vivo. Here we report a
doxorubicinꢀloaded photosensitizerꢀcore pHꢀresponsive copolymer nanocarrier with
high tumor targeting and anticancer effects due to integration of PDT with
chemotherapy. The pHꢀresponsive photosensitizerꢀcore fourꢀarmed starꢀshaped
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copolymer,
methacrylate)ꢀpoly(εꢀcaprolactone)]4ꢀzinc
benzyloxyl)phthalocyanine
[methoxyꢀpoly(ethylene
glycol)ꢀpoly(2ꢀ(N,Nꢀdiethylamino)ethyl
βꢀtetraꢀ(4ꢀcarboxyl
(PDCZP), was prepared, which was a molecular
spherical nanocarrier in aqueous media. The carriers changed from small at high pH
to large at low pH (51, 105, and 342 nm at pH 7.4, 6.5, and 5.0, respectively) and the
zeta potential gradually increased (7.15, 16.2, and 26.1 mV at the above pH,
respectively). PDCZP had a longer emission wavelength (max. 677 nm) than the
parent photosensitizer, favoring light penetration through biological tissues. The
singlet oxygen (1O2) quantum yield of PDCZP was 0.41. Doxorubicin (DOX) showed
rapid release from PDCZP in the acidic media. More importantly, the drugꢀloaded
nanocarrier showed the better in vitro and in vivo anticancer effects under lighting on
MCFꢀ7, SW480 cells and HepG2 cells and the murine hepatocellular carcinoma H22
models than the other groups. PDCZP showed a high tumor targeting effect based on
the enhanced permeation and retention effect and its small size. The
photosensitizerꢀcore nanocarrier is a promising photodynamic nanocarrier for
integrating other therapies.
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KEYWORDS: doxorubicin, pHꢀresponsive, photodynamic therapy, phthalocyanine,
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photosensitizer, tumor targeting
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INTRODUCTION
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Photodynamic therapy (PDT) is an emerging noninvasive approach to skin and
cavity diseases, and some malignant tumors.1ꢀ3 In PDT, lightꢀactivating
photosensitizers can pass on their excess energy to surrounding molecular oxygen to
produce reactive oxygen species (ROS), such as free radicals and singlet oxygen (1O2)
that are toxic to cells and tissues.4ꢀ6 Generally, the major administration route of
photosensitizers is intravenous (i.v.) injection. However, the clinically applied
systemic photosensitizers combining with the longꢀterm exposure to light radiation
could lead to severe side effects, such as hepatotoxicity, renal toxicity, hypertension,
hyperlipidemia, and skin cancers.7
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Anticancer nanocarriers are fascinating due to their tumor targeting and
environmental triggered drug release.8 Nanocarriers are also introduced to deliver
anticancer photosensitizers to obtain tumor targeting.9ꢀ13 Furthermore, scientists are
trying to combine chemotherapy and PDT for the treatment of cancer. Generally, a
nanocarrier is used for entrapping both anticancer agents and photosensitizers.14ꢀ16
Additionally, anticancer agents were covalently conjugated with photosensitizers,
such as the conjugate of sulfonated aluminum phthalocyanine and doxorubicin
(DOX),17 and the conjugate of silicon phthalocyanine and paclitaxel.18
As a matter of fact, a photosensitizer does not need to be released into the
surroundings because it is only the resource of singlet oxygen under lighting. Here,
we designed a novel photosensitizerꢀcore polymer as the drugꢀloaded nanocarrier, i.e.,
the photosensitizer is one part of the nanocarrier. Moreover, some functional groups
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are induced into the polymer. The novel functional copolymer consists of zinc
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phthalocyanine as the core and
a
sequentially conjugated hydrophobic
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chain/pHꢀresponsive chain/hydrophilic chain to form a fourꢀarmed starꢀshaped
copolymer (Figure 1). When a drug (e.g., DOX) is entrapped in this copolymer, we
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call it
a
doxorubicinꢀloaded photosensitizerꢀcore pHꢀresponsive copolymer
nanocarrier. The copolymer itself is a photosensitizer for PDT, and the entrapped
DOX is released in a pHꢀdependent manner after targeting tumors (Figure 1). Our
previous research has showed
a
drugꢀloaded starꢀshaped pHꢀresponsive
monomolecular copolymer nanocarriers for tumor targeting and cancer therapy.19 We
demonstrated
that
the
tetraꢀ(methoxyꢀpoly(ethylene
glycol)ꢀpoly(2ꢀ(N,Nꢀdiethylamino)ethyl
methacrylate)ꢀpoly(εꢀcaprolactone)
pentaerythritol ((mPEGꢀpDEAꢀPCL)4ꢀPET, PDCP) nanocarriers could be used to
deliver hydrophobic anticancer drugs for cancer therapy with tumor targeting and
controlled drug release. In this study, we replaced the pentaerythritol core with a zinc
phthalocyanine core that is a photosensitizer while using the modified copolymer to
load an anticancer drug.
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pH 7.4
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Drugs
Lighting
1O2
pH 6.5
Figure 1. Molecular structure of PDCZP and illustration of its pHꢀdependent drug
release and combined therapy of chemotherapy and photodynamic therapy.
EXPERIMENTAL SECTION
Materials. Doxorubicin (DOX) chloride was a gift from Zhejiang Hisun
Pharmaceutical Co., Ltd., China. εꢀCaprolactone (CL) was purchased from Aladdin,
China. Tinoctoate (Sn(Oct)2) was purchased from Sigma. Organic solvents were of
analytical grade and other chemicals were of reagent grade. 9,10ꢀDimethylanthracene
(DMA) was from TCI (Tokyo, Japan). Zinc phthalocyanine (ZnPc) from TCI was
used as the control in the determination of singlet oxygen quantum yield. Purified
water was prepared using Heal Force Super NW Water System (Shanghai Canrex
Analytic Instrument Co., Ltd., Shanghai, China). The molecular weight cutꢀoff of
dialysis bags was from Union Carbide Corporation, New Jersey, USA. Ultraviolet–
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visible (UV–vis) spectra, infrared (IR) spectra, and nuclear magnetic resonance
(NMR) spectra were recorded on a Purkinje TUꢀ1901 spectrophotometer (Beijing
Purkinje General Instrument Co., Ltd., Beijing, China), a BioꢀRad FTSꢀ65A infrared
ray spectrometer, and a JNMꢀECAꢀ400 NMR spectrometer, respectively.
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Animals. Healthy female Kunming mice (18-22 g) were from the Laboratory
Animal Center of Beijing Institute of Radiation Medicine (BIRM). The animals were
handled and surgically treated in strictly accordance with the Guidelines for the Use
of Laboratory Animals. All the studies were conducted in accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory animals. The
animal experiments were approved by the Animal Care Committee of BIRM.
Synthesis of PDCZP. The synthetic route of (mPEGꢀpDEAꢀPCL)4ꢀZnPC4
(PDCZP) is shown in Figure 2. mPEG was methoxyꢀpoly(ethylene glycol) 2000
(Acros, Belgium). mPEGꢀpDEAꢀN3 was synthesized in our lab according to our
previous research.20 The propargylꢀterminated PCL was obtained by ringꢀopening
polymerization of εꢀCL under the coꢀinitiation of propargyl alcohol and Sn(Oct)2.21
The polymerization was performed in a glovebox with a water content of less than 0.1
ppm. Propargyl alcohol (1.15 ml, 0.02 mol) and εꢀCL (21.3 ml, 0.2 mol) were added
to freshly dried toluene (20 ml) in a flask. Sn(Oct)2 (0.12 g) was added and the
solution was stirred at 80 °C for 12 h. The mixture was concentrated under vacuum.
After precipitation in cold diethyl ether, the polymer was obtained and dried under
vacuum overnight. Click reaction was used to prepare PDC.22 The
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propargylꢀterminated PCL (1 g) and mPEGꢀpDEAꢀN3 (1.5 g) were dissolved in
tetrahydrofuran (THF, 20 ml). CuI (0.005 g) and 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene
(DBU) (0.18 ml) were added. The solution was stirred in a nitrogenꢀfilled sealed flask
at 60 °C for 24 h before being passed through a neutral alumina column to remove the
copper catalyst. The solvent was removed to obtain a viscous liquid that was then
dialyzed against water using a dialysis bag (cutꢀoff MW, 7000) for 72 h. The solution
in the bag was lyophilized to obtain a white solid of PDC. Infrared IR (KBr) νmax
(cmꢀ1): 2967, 2872 (ꢀCH2ꢀ), 1726 (ꢀC=Oꢀ), 1113 (ꢀCꢀOꢀCꢀ), 1461 (ꢀCH2ꢀ). The
absorption peak of azide group disappeared completely after the click reaction.
Meanwhile, a strong absorption at 1726 cmꢀ1 contributing to the C=O stretching of
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PCL block was observed. H NMR (DMSOꢀd6, 400 MHz) δ (ppm): 3.66 (pDEA,
ꢀCH2ꢀOꢀ), 2.65 (pDEA, ꢀCH2ꢀNꢀ), 4.0 (mPEG, ꢀCH2ꢀ), 3.38 (mPEG, CH3ꢀOꢀ), 2.35
(PCL，ꢀCH2ꢀ), 3.65 (PCL, –OH), 5.32 (ꢀCH2OꢀCOꢀ), 7.80 (ꢀNCHꢀ).
The possible polymerization degrees in the molecule of mPEGmꢀpDEAnꢀPCLl
(PDC) may be accessed from the comprehensive structural information. The
MALDIꢀTOF mass spectrum of mPEGꢀpDEAꢀBr showed the peaks of 2347, 4105,
and 6055 m/z (Figure S1 in the Supporting Information), which belonged to mPEG,
pDEA, and mPEGꢀpDEAꢀBr, respectively. According to the molecular structure, the
polymerization degree of PEG, i.e., m, was about 53, and that of pDEA, i.e., n, was
1
about 12. According to the H NMR spectrum of propargylꢀterminated PCL (Figure
S2), the peak integral of 2ꢀsite H (δ, 2.31 ppm) of PCL units was 19 times of that of
propargyl H (δ, 4.68 ppm), indicating that the polymerization degree of PCL was
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about 19. Therefore, the polymerization degrees in mPEGꢀpDEAꢀPCL were possibly
53 (m), 12 (n), and 19 (l), respectively.
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ZnPC4 was prepared according to our previous research.23 ZnPC4 (1.13 g, 1 mmol)
and PDC (5.03 g) were dissolved in THF (15 ml) by turns. DCC (0.307 g, 1.5 mmol)
and N,N’ꢀdimethylaminopyridine (DMAP, 0.121 g, 0.99 mmol) were added and then
agitated at room temperature for 48 h. The solution was evaporated under vacuum
until ca. 1 ml and then purified on a silica column with THF as the eluent. The green
filtrate was dialyzed against water using a dialysis bag (cutꢀoff MW, 8000ꢀ14000) for
72 h and refreshing water every 3 h. The solution in the bag was lyophilized to obtain
a green solid of PDCZP. UVꢀvis (THF): λmax = 348, 681 nm; IR (KBr) νmax (cmꢀ1):
732.1 (ZnꢀN), 1088.4, 1504.5, 1601.1, 1627.0 (phthalocyanine), 1241.6 (ArꢀOꢀAr),
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1726.8 (ꢀCOꢀ), 2853.3 (ꢀCH2ꢀ); H NMR (DMSOꢀd6, 400 MHz) δ (ppm): 1.5 (m,
PcꢀPc), 2.25 (m, PCL, ꢀCH2ꢀ), 4.0 (m, PEG, ꢀCH2ꢀ), 3.5 (m, pDEA, ꢀCH2ꢀOꢀ), 2.5 (m,
pDEA, ꢀCH2ꢀNꢀ), 5.3 (d, ꢀCH2ꢀCOꢀ), 7.0ꢀ7.8 (m, Hꢀphthalocyanine), 8.0 (m, HꢀAr).
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O
(1)
HO
COOH
OH
K2CO3
DMSO
NC
NC
NO2
ZnCl2/DBU
DMF
O
CN
CN
OH
+
O
N
HOOC
O
N
O
N
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8
Zn
N
N
N
9
O
N
N
O
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O
OH
OH
ZnPC4
O
(2)
CH3
O
CH3
TEA
Br
Br
H(OCH2CH2)mOCH3
mPEG
Br
O CH2CH2)mOCH3
+
H3C
H3C
O
mPEG-Br
DEA
CuBr, bpy
O
O
N3
Br
CH3O(CH2CH2
O
CH3O(CH2CH2
O
n
m
n
NaN3
m
O
O
O
O
mPEG-pDEA-N3
mPEG-pDEA-Br
N
N
O
N
H
l
HC
O
O
O
N
N
CH3O(CH2CH2
O
n
m
O
H
O
l
O
O
O
N
PDC
(3)
O
mPEG-pDEA-PCL
O
O
N
O
N
N
DMAP/DCC
ZnPC4
PDC
Zn
N
+
N
N
N
O
N
O
O
PCL-pDEA-mPEG
O
PDCZP
Figure 2. Synthetic routes of PDCZP.
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Preparation and Characterization of PDCZP Nanocarriers. A PDCZP solution
(2 mg/ml) in THF was prepared. The solution was injected into water using a
microsyringe under bath sonication until a homogenous green suspension was
obtained. THF was removed from the suspension under vacuum at 37 °C for 5 min
followed by evaporation at room temperature. The DOXꢀloaded PDCZP nanocarriers
were prepared using a similar method to the one mentioned above. DOX chloride was
first neutralized with triethylamine (1:3, mol/mol) in a little acetone and then mixed
with a PDCZP solution in THF at a series of DOX/PDCZP ratios (1:2, 1:5, 1:10, 1:20,
w/w). The mixed solutions were separately injected in water followed by removal of
solvents as mentioned above to obtain a homogenous, nearly transparent, and green
suspension that could be further concentrated following removal of some water or
directly lyophilized to powders.
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The THF solutions of ZnPc, ZnPC4, and PDCZP were UVꢀvis scanned in the range
of 200 to 800 nm, respectively. PDCZP nanocarriers were observed under a Hitachi
Hꢀ7650 80ꢀkV transmission electron microscope (TEM) after staining with a sodium
phosphotungstate solution (pH 7.4) as reported previously.24 A dynamic lighting
scattering (DLS) method on Zetasizer Nano ZS (Malvern, UK) was applied to
measure the sizes of PDCZP nanocarriers at different pH. The zeta potentials of
PDCP nanocarriers were also measured with the above instrument at 25 °C.
Determination of DOX. The DOXꢀloaded PDCZP nanocarriers were separated
from the suspension by centrifuging through an ultrafiltration tube (cutꢀoff MW,
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10000, PALL Corporation, USA) at 5000× g for 10 min. The nanocarriers remained
above the ultrafiltration membrane, while free DOX existed in the filtrate. DOX was
analyzed using the high performance liquid chromatography (HPLC), involving a
system of LCꢀ10Avp series (Shimadzu, Japan), a Tianhe® ODSꢀC18 column (150 mm
× 4.6 mm, 10 ꢁm) at 30 °C, a mobile phase of methanol/20 mM ammonium
acetate/acetic acid (60:40:5.4, v/v) with a flow rate of 0.8 ml/min, and a detection
wavelength of 254 nm. The drug encapsulation efficiency (EE) and drug loading
efficiency (DL) were calculated as Eq. (1) and (2).
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WD1
EE =
DL =
×100%
WD2
(1)
(2)
WD1
×100%
WPDPZP +WD1
where WD1 was the DOX content in the nanocarriers, WD2 was the total added drug
content, WPDCZP was the added copolymer content.
Drug Release from PDCZP Nanocarriers. A dialysis method was used to
evaluate drug release from PDCZP nanocarriers. The DOXꢀloaded PDCZP
suspension (2 ml) was put into a dialysis bag (cutꢀoff MW, 3500) and dialyzed against
the media (50 ml) in an oscillating incubator (120 rpm, 37 °C). Aliquots (1 ml) of the
buffered solutions were collected at the predetermined time points and then
supplemented with the same volume of media. The released drug was determined
using the above HPLC method and the cumulative release was calculated. The
dialysis media were phosphate buffered solutions (PBS, pH 5.0, 6.5, or 7.4).
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Determination of Singlet Oxygen Quantum Yield of PDCZP. Determination of
singlet oxygen quantum yield was performed according to our previous report.23 The
test was based on the changes of a singlet oxygen chemical quencher DMA in DMF.
The stock solutions of ZnPc, DMA and PDCZP in DMF were prepared. The solutions
of ZnPc (3 ꢁM), PDCZP (3 ꢁM), DMA (90 ꢁM), ZnPc (3 ꢁM)/DMA (90 ꢁM),
PDCZP (3 ꢁM)/DMA (90 ꢁM) were obtained after dilution and/or mixing of the stock
solutions with DMF. A 100ꢀmW 657ꢀnm laser emitter (YSHINELASER, China) was
used to excite the solutions (each aliquot of 3 ml) of photosensitizers in the 1ꢀcm
cuvette through a 9ꢀmm diameter hole. The laser radiation process was performed
under dark conditions. The light intensity was determined as 8.12 mW/cm2 with Nova
II (OPHIR, Israel). The absorbance of the radiated solutions at 405 nm was measured
on the UVꢀvis spectrophotometer every 30 s. The eliminated DMA rate constants
(KDMA) were obtained as Eq. (3).
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A0
ln
= KDMAt
At
(3)
where A0 was the initial absorbance of DMAꢀcontained solutions without laser
radiation, while At was the absorbance of solutions at t time. The absorbance of DMA
solutions was also measured in the same time range as above to confirm that DMA
was not oxidized by the oxygen in the atmosphere. DMA oxidation was used to
determine the singlet molecular oxygen (1O2) produced by the photosensitizers.
The photon absorption rate constants (Kpho) of photosensitizers were calculated
using Eq. (4).
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0.97× P×(1−10−AL
(0.1197/λ)×V
)
Kpho
=
(4)
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where P was the light intensity (8.12 mW/cm2), A was the absorbance of
photosensitizer solutions at the laser wavelength (657 nm), V was the volume of
solutions (3 ml), L was the light length on the solutions (1 cm), λ was the light length
(657 nm), and the value of 0.97 was the correct factor of the reflecting light at the
interface of air/glass. Singlet oxygen quantum yields (Φ) of photosensitizers were the
direct ratios of DMA oxidation rate. The ΦZnPc of ZnPc in DMF was 0.56 25. Therefore,
the ΦPDCZP can be calculated as Eq. (5).
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(KDMA / Kpho
)
PDCZP
ΦPDCZP
ΦZnPc
=
(KDMA / Kpho
)
ZnPc
(5)
Cytotoxicity Test. DOX chloride solutions, PDCZP suspensions, and DOXꢀloaded
PDCZP suspensions were sterilized with 60Co γꢀray radiation. Cytotoxic experiments
were performed on human breast cancer MCFꢀ7 cells, human colon cancer SW480
cells, and human hepatocellular cancer HepG2 cells. The cells were cultured in a
DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C. The cells were
seeded in the 96ꢀwell plates with 2000 cells/well to adhere to the plates thoroughly.
Four hours after incubation in the dark, the plates were exposed under a fluorescent
lamp with the light intensity of 4500 Lx for 30 min, and then incubated for 24 h.
Aliquots (20 ꢁl) of MTT solutions were separately added to each well and the cells
were further incubated for 5 h. The media were subsequently collected and dimethyl
sulphoxide (DMSO, 150 ꢁl) was added to dissolve the darkꢀblue formazan crystals.
Formazan was measured at 490 nm using a microplate reader (Multiskan MK3，
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Thermo Scientific, US). Cell viability was calculated as (absorbance of experimental
groups)/(absorbance of negative control)×100%. The half concentration of inhibition
(IC50) was then deduced.
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Hemolysis Assay. Rat erythrocyte suspensions (2%, v/v) in saline were prepared
according to the report.26 A series of samples were prepared, containing 1%
erythrocytes and a series of PDCZP suspensions in saline and they were incubated at
37 °C until observation. Hemolysis was observed with naked eyes against the
completely hemolytic sample of waterꢀmixed erythrocytes. Four hours later, the
supernatants were also collected and measured with the spectrophotometry at 577 nm.
Tumor Implantation. A murine hepatoma H22 cancer cell line was used. H22
cancer cells were extracted from the abdominal dropsy of mice bearing cancer cells
and diluted with saline to achieve a concentration of 3×107 cells/ml. Aliquots (0.2 ml)
of the cell suspensions were separately and subcutaneously (s.c.) injected into the
mice under the right forelimb. Five days after transplant, the injected mice of
appropriate tumor volumes were selected for tissue distribution and
pharmacodynamic studies.
Pharmacodynamic Study. Thirty mice with the expected tumor volume were
equally divided into six groups: (A) the saline group treated with saline; (B) the blank
PDCZP group treated with PDCZP/dark; (C) the light group just with lighting alone;
(D) the PDCZP/light group treated with PDCZP/lighting; (E) the DOXꢀloaded
PDCZP group treated with DOXꢀloaded PDCZP/dark; (F) the DOXꢀloaded
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PDCZP/light group treated with DOXꢀloaded PDCZP/lighting. PDCZP solution had
0.68 mg PDCZP/ml. DOXꢀloaded PDCZP solution had 207 ꢁM DOX and 0.78 mg
PDCZP/ml. The dose was 3 mg DOX/kg for Group E and F, and 17 mg PDCZP/kg
for Group B and D. Aliquots (0.5 ml) of the medicines were i.v. injected to the mice
via the tail veins once every two days totally for 3 times. All the animals were housed
under dark conditions unless they were treated with a laser light. After injection, the
mice were raised for 12 h under dark conditions and the groups of C, D, and F were
lighted on the tumor xenografts for 15 min using a large HeꢀNe laser lighter (JKꢀ3,
equipped in BIRM).
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Xenograft sizes were measured in two perpendicular dimensions using a caliper
once a day after treatment. The first administration day was recorded as Day 1. Tumor
volume (V) was calculated as 0.5L×W2, where L was the largest superficial diameter,
and W was the smallest superficial diameter of the xenograft. Six 6 days later, the
mice were sacrificed. Tumors were taken and weighed to calculate the tumor
inhibitory rates as Eq. (6).
Wblank −Wtest
Tumor inhibitory rate (%) =
×100%
Wblank
(6)
where Wblank and Wtest were the average tumor weight of the control group and the test
groups, respectively. Mice were weighed daily.
All the results were expressed as mean±standard deviation (SD). Student’s tꢀtest
was used for comparison of the two means. A significance level of p < 0.05 and p <
0.01 represented statistical difference and significant difference, respectively.
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Fluorescence Non-Invasively Tumor Imaging. A DOXꢀloaded PDCZP solution
of 207 ꢁM DOX and 0.78 mg PDCZP/ml was used. It was i.v. injected into the H22
tumorꢀbearing mice via tail veins at the dose of DOX 10 mg/kg. The mice were
sacrificed. The organs, including the heart, liver, spleen, lung, kidney, and tumor,
were immediately surgically isolated at 0.5, 1, 2, 4 and 8 h after administration,
respectively. The organs were washed with saline and immersed in formalin before
imaging. The organs were moved to the imaging chamber for scanning. Excitation
and emission spots were rasterꢀscanned in 1 mm steps to generate emission
wavelength scans. A 675 nm pulsed laser diode was used to excite PDCZP without
exciting the tissue matrix and fluorescence interference. The organs were imaged on
an imaging system (NightOWL II LB 983, Germany) and a cold CCD
fluorescence/illuminating tissues imaging system (LB983ꢀNC100, Berphold,
Germany).
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RESULTS AND DISCUSSION
Formation of the Drug-Loaded Photosensitizer-Core Nanocarrier. The
preparation of the nanocarriers was simple, i.e., injecting its THF solution into water
under bath sonication. THF was a good solvent of PDCZP and easily removed under
vacuum, which was why it was selected. In this study, the loaded drug, i.e., DOX, was
easily entrapped into the large intraꢀspace of PDCZP nanocarriers using the similar
process as above, wherein the alkalized DOX was dissolved in THF with PDCZP.
The 1:10 (w/w) ratio of DOX/PDCZP was used after screening. The drug
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encapsulation efficiency (EE) and drug loading efficiency (DL) were 94.3% and
14.46%, respectively. A homogenously transparent green solution was obtained.
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Properties of the Nanocarriers. PDCZP nanocarriers showed different sizes and
zeta potentials depending on pH (Figure 3A-D). The carriers changed from small at
high pH to large at low pH with the size of 51, 105, and 342 nm at pH 7.4, 6.5, and
5.0, respectively. The zeta potential was gradually increased from 7.15, 16.2, to 26.1
mV at the above pH, respectively. The TEM images also showed a similar result
(Figure 3A-C). The >300 nm sizes and the significant size changes indicated the
supramolecular assemblies of PDCZP could be formed in the nanocarriers. However,
the strong pH dependency of PDCZP nanocarriers was confirmed. Because of the
small size, PDCZP nanocarriers could easily enter tumor tissues from the circulation
based on the enhanced permeability and retention (EPR) effect.27 PDCZP nanocarriers
might be stable because no significant precipitates appeared within one month. The
outside PEG chains of PDCZP could produce the space static effect to prevent the
aggregation of nanocarriers.28
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Figure 3. Properties of PDCZP nanocarriers. TEM images at pH 5.0 (A), pH 6.5 (B),
and pH 7.4 (C). Size and zeta potential profiles at different pH (D). DOX release
profiles of DOXꢀloaded PDCZP at different pH (E).
PDCZP and ZnPC4 showed two UVꢀvis absorption peaks at 354 nm and 677 nm.
The conventional phthalocyanine, i.e., ZnPc, has two absorption peaks at 342 nm and
669 nm (Figure S3). The red shifts of PDCZP and ZnPC4 absorption may be attributed
to the fourꢀcarboxyl derivation of ZnPc. More importantly, the light of long
wavelengths can penetrate through biological tissues into the deep sites better than the
light of short wavelengths.30
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PDCZP did not show hemolysis within 3 h after incubation with erythrocytes even
when PDCZP concentration was up to 5 mg/ml. Moreover, four hours postꢀincubation,
the supernatants of a series of samples did not show hemolysis because the
absorbances were close to zero. Therefore, the i.v. injected PDCZP was safe.
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pH-Dependent Drug Release from the Nanocarriers. PDCZP is a pHꢀresponsive
nanocarrier, resulting from the pHꢀresponsive pDEA chain. pDEA is
poly(2ꢀ(N,Nꢀdiethylamino)ethyl methacrylate), occupying one tertiary amine group in
its monomer. The pDEA chains could shrink in weakly basic environments (such as
pH 7.4 in the circulation) due to the hydrophobic form. However, the chains could
extend in weakly acidic environments (such as pH 6.5 in the tumor microenvironment
or 5.0 in the endosomes) because the amines of pDEA transformed into the protonized
forms that were hydrophilic. Our previous research on the pDEAꢀcomposed
coꢀpolymers also showed similar results.19, 20, 31 The pHꢀresponsive function of
PDCZP could make it open at low pH and close at high pH. Therefore, simply
switching the pH of media can be used for entrapping or releasing the cargos in
PDCZP. In this study, the release of DOX from the PDCZP nanocarriers was
significantly pHꢀdependent and was accelerated as pH decreased (Figure 3B).
Furthermore, we predicted that DOX would remain in PDCZP nanocarriers when
circulating in the blood (pH 7.4), and release itself when reaching tumor tissues or
entering the endosomes.19
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Photodynamic Effect of Nanocarriers. Singlet oxygen quantum yield (Φ) is a
key parameter of photosensitizers. High Φ values indicate high production of ROS.
The Φ of PDCZP was 0.41, a little lower than the values of parent ZnPc (0.56) and
another amphiphilic derivative of ZnPc (ZnPcꢀBrij 58 conjugate, ZPB, 0.64)
previously prepared in our lab.23 The long PDC chains of PDCZP might shield energy
transportation or oxygen diffusion, leading to decrease of the Φ value.
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Cytotoxicity of DOX-Loaded Nanocarrier. Three types of cancer cell lines were
used for detecting the in vitro anticancer effects of DOX, PDCZP, and DOXꢀloaded
PDCZP. Cell viabilities were measured following addition of different medicines,
respectively, and a 30ꢀmin lighting process with a fluorescent lamp. We found that
lighting played a key role in the anticancer effect of PDCZP used alone or
DOXꢀloaded PDCZP (Figure 4). The group of DOXꢀloaded PDCZP/light had a
highest cytotoxicity than the other two groups, and the difference was statistically
significant (p < 0.01). In addition, PDCZP did not show any cytotoxic effect on a
melanoma cell line B16ꢀF10 under dark conditions even at the high concentration of
100 ꢁg/ml (Figure S4). Therefore, the photodynamic effect of PDCZP carriers was
confirmed.
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Figure 4. Cytotoxicity of DOX, PDCZP, and DOXꢀloaded PDCZP on the cancer cell
lines, including HepG2, MCFꢀ7, and SW480, respectively. “Low”, “Middle”, and
“High” indicate the concentration levels of DOX (13.0, 31.1, 60.3 ꢁM), PDCZP (40,
100, 200 ꢁg/ml), and DOXꢀloaded PDCZP (DOX at 13.2, 31.8, 63.6 ꢁM, and PDCZP
at 50, 120, 240 ꢁg/ml), respectively. All the data are presented as means ± SD (n=6).
Anticancer Effect of DOX-Loaded Nanocarrier. The murine hepatocellular
carcinoma (H22)ꢀbearing mice were i.v. administrated with saline, PDCZP, and
DOXꢀloaded PDCZP, respectively, once every two days. Twelve hours after injection,
the mice were exposed to laser lighting for 15 min or chronically under dark
conditions. On the second day after the final administration, the mice were sacrificed.
In this study, free DOX solutions were also injected to the mice which, however,
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showed little movement, dull hair, and twitch from Day 3. Finally, some mice died
from the serious side effect, which should be attributed to the strong heart toxicity of
free DOX.31 In contrast, no mice died in the other groups. It is known that free DOX
shows strong heart toxicity.32 In our previous research, free DOX solutions were also
demonstrated to have serious toxicity on healthy mice. However, DOXꢀloaded
nanocarriers decreased the toxicity due to the low distribution in the heart.9 Therefore,
a nanoscale formulation, i.e., liposomal DOX, becomes a major alternative of DOX
solutions in clinics.
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Both PDCZP/light and DOXꢀloaded PDCZP/light showed good anticancer effects
according to the tumor volume (Figure 5A). Furthermore, the group of DOXꢀloaded
PDCZP/light had the lowest mean tumor weight. Interestingly, the dark/light groups
of the same medicines showed statistical differences in tumor weight with
p < 0.05
(PDCZP under dark vs. lighting) and < 0.01 (DOXꢀloaded PDCZP under dark vs.
p
lighting). The tumor inhibitory rate was 67.60% in the DOXꢀloaded PDCZP/light
group, but was 27.77%, 54.92%, and 28.06% in the other groups, including the
PDCZP/dark group, the PDCZP/light group, and the DOXꢀloaded PDCZP/dark
group. We noted that tumor inhibition was mild in the PDCZP/dark group. PDCZP is
a large copolymer, composed of mPEG, pDEA, PCL, ZnPC4 moieties, where ester
bonds dominate. Therefore, it would finally be degraded in vivo and excreted to urine
though the process of degradation might be slow. Some unknown mechanisms might
have led to the systemic toxicity because the animal body weight of the PDCZP/dark
group decreased, which further led to tumor weight decrease. The DOXꢀPDCZP/dark
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group also showed a similar result. Fortunately, the DOXꢀPDCZP/light group showed
smaller tumors and higher body weight. Cancer is a complicated disease where tumor
growth, body weight, and systemic toxicity are mutually related. Therefore, the
combinatorial therapy using the DOXꢀloaded photosensitizerꢀcore nanocarriers could
be a perfect method with high anticancer effect and very weak side effect.
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Figure 5. Anticancer effects of different medicines under dark conditions or lighting
on the H22 tumorꢀbearing mice. Tumor volume profiles (A). Body weight profiles
(B). Tumor weight after treatments (C). * p < 0.05; ** p < 0.01. (D) Tumors after
treatments. All the data are presented as means ± SD (n=5).
Tumor Targeting of the Nanocarriers. We detected the tumor targeting effect of
PDCZP using the fluorescence nonꢀinvasively tumor imaging method. Besides the
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photodynamic effect, PDCZP has the fluorescent function for imaging in vivo. The
imaging results showed that the distribution of DOXꢀloaded PDCZP into the organs
was slow, which may be attributed to the longꢀcirculating effect of PDCZP due to its
long hydrophilic chains (Figure 6). Two hours postꢀadministration, PDCZP reached
the maximal level in the liver. And then, PDCZP in the liver decreased and increased
in the heart, kidney and tumor. Eight hours later, PDCZP reached the maximal levels
in the kidney and tumor. Hence, the tumor targeting of PDCZP was confirmed.
PDCZP or its metabolites could be eliminated from the major distributed tissues (e.g,
the liver) to the blood. And they would finally be excreted into urine. Cationic small
nanoparticles are also readily excreted into urine after injection.33 Therefore, PDCZP
could not accumulate in the organs, and only to be eliminated from the body.
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Figure 6. Fluorescence nonꢀinvasively tumor imaging after injecting the DOXꢀloaded
PDCZP to the tumorꢀbearing mice.
CONCLUSIONS
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A doxorubicinꢀloaded photosensitizerꢀcore pHꢀresponsive starꢀshaped copolymer
nanocarrier was prepared in this study. The unique advantage of this nanocarrier is the
simultaneous work of chemotherapy and PDT. We demonstrated that one case of this
functional nanocarrier, i.e., the DOXꢀloaded PDCZP, showed pHꢀresponsive drug
release, high anticancer effect, and tumor targeting effect. The photosensitizerꢀcore
nanocarrier is a promising photodynamic nanocarrier that can be integrated with other
therapies.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI: 10.1021/
MALDIꢀTOF mass spectrum of mPEGꢀpDEAꢀBr, 1H NMR spectrum of
propargylꢀterminated PCL, UV spectra of ZnPc, ZnPC4, and PDCZP, cell viability of
PDCZP on B16ꢀF10 cells under dark conditions.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: jinyg@sina.com
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
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We are grateful for the support from the National Natural Science Foundation of
China (No. 81373340). We thank Prof. Shoujun Yuan for providing all the cancer cell
lines, and Prof. Xiansheng Lu for Language editing and proofꢀreading.
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