Leveraging a polycationic polymer to direct tunable loading of an anticancer agent and photosensitizer with opposite charges for chemo-photodynamic therapy

Article abstract of DOI:10.1039/c9tb02400j

Herein, we reported a primary amine containing polycationic polymer to load an oppositely charged anticancer drug (doxorubicin, DOX) and a photosensitizer (chlorin e6, Ce6) for combinational chemo-photodynamic therapy. The electrostatic interactions as well as other multiple interactions between the polymer and payloads endowed the drug-loaded nanoparticles with excellent stability. Moreover, the electrostatic attraction between the cationic polymer and anionic Ce6 dictated that Ce6 had higher loading efficiency than DOX. DOX showed pH-responsive drug release owing to the increased solubility of protonated DOX and reduced interaction with the partially protonated polymer under acidic conditions. In contrast, Ce6 showed pH-insensitive release because of the smaller change in solubility and the intense interactions between Ce6 and the polymer. Synergistic chemo/photodynamic therapy of 4T1 cancer cells was achieved by light-triggered reactive oxygen species (ROS)-mediated enhanced cellular uptake and effective endo/lysosomal escape of drug-loaded nanoparticles. Our study demonstrated that the polycationic polymer could act as a robust carrier for differential loading and release of oppositely charged cargos for combinational therapy.

Full text of DOI:10.1039/c9tb02400j

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X. Peng, B. Zhang, Q. Pan, S. Li, B. He and Y. Pu, J. Mater. Chem. B, 2020, DOI: 10.1039/C9TB02400J.
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ISSN 2050-750X
PAPER
Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
particles
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DOI: 10.1039/C9TB02400J
Journal Name
ARTICLE
Leveraging a polycationic polymer to direct tunable loading
of anticancer agent and photosensitizer with opposite
charges for chemo-photodynamic therapy
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
b
b
b
a*
b
Mingying Zhao , Shiyu Wan , Xinyu Peng , Boya Zhang , Qingqing Pan , Sai Li , Bin He , and Yuji
DOI: 10.1039/x0xx00000x
b*
Pu
www.rsc.org/
Herein, we ported a primary containing polycationic polymer to load oppositely-charged anticancer drug (doxorubicin, DOX)
and photosensitizer (chlorin e6, Ce6) for combinational chemo-photodynamic therapy. The electrostatic interactions as well
as other multiple interactions between polymer and payloads rendered the drug-loaded nanoparticles excellent stability.
Moreover, the electrostatic attraction between cationic polymer and anionic Ce6 dictated that Ce6 had higher loading
efficiency than DOX. DOX showed a pH-responsive drug release owing to the elevated solubility of protonated DOX and
reduced interaction with partially protonated polymer in acidic condition. In contrast, Ce6 showed pH-insensitive release
because of the less change of solubility and the intense interactions between Ce6 and polymer. Synergistic
chemo/photodynamic therapy of 4T1 cancer cells was achieved by light-triggered reactive oxygen species (ROS)-mediated
enhanced cellular uptake and effective endo/lysosomal escape of drug-loaded nanoparticles. Our study demonstrated that
the polycationic polymers could act as a robust polymer carrier for differential loading and release of oppositely-charged
cargos for a combinational therapy.
hydrophobic interactions, improving the anticancer efficacy and
7
-12
Introduction
reducing side effects.
However, many anticancer drugs and
photosensitizers also have amine and/or carboxylic groups,
which are partly positively and negatively charged in neutral pH.
For example, doxorubicin (DOX) and chlorin e6 (Ce6) are a
typical anticancer drug and PS, respectively. They are both
hydrophobic because of the aromatic structure; meanwhile,
DOX and Ce6 have amine and carboxylic groups, respectively. In
a typical DDS, DOX and Ce6 could be simultaneously loaded into
an amphiphilic block copolymer with no charge to generate
Chemotherapy is one of the main strategies for clinical
treatment of cancer. However, the chemotherapeutics’ poor
killing selectivity between cancer and normal cells inevitably
leads to severe systemic toxicity in chemotherapy.
Photodynamic therapy (PDT), another approved cancer
treatment modality, kills cancer cells by reactive oxygen species
1
(ROS) generated from photosensitizers (PSs) after light
irradiation and has garnered much attention due to the
inherent noninvasive and spatiotemporally controllable
nature.2 Owing to the different anticancer mechanisms of
chemotherapy and PDT, the chemo-photodynamic combination
therapy has emerged as a strategy of great potential in
efficiently ablating tumors and alleviating systemic toxicity
1
3-18
polymeric nanoparticles.
Recently, Zhang et al. reported
that DOX and Ce6 could be directly used to construct carrier-
free nanoparticles by the multiple interactions between DOX
, 3
1
9
and Ce6 for chemo-photodynamic therapy. Such nanodrugs
could exhibit very high drug encapsulation efficiency and drug
feeding molar ratio-dependent sizes. However, DOX and Ce6
showed comparable and relatively rapid drug release behaviors
4
-6
caused by traditional chemotherapy. However, the small and
hydrophobic nature of most of the chemotherapeutics and PSs
has severely limited their bioavailability for the insolubility in
water.
(about 60%–80% DOX and Ce6 were released within 48 h),
which might lead to potential drug leakage in blood circulation
and thereby systemic toxicity.
Inspired by the hydrophobic nature of chemotherapeutics
and PSs, various drug delivery systems (DDSs) have been
developed to encapsulate chemotherapeutics and/or PSs by
Polycationic polymers, natural or synthetic, have recently
attracted wide attention and widely applied in many biomedical
fields. For example, many natural antimicrobial peptides, widely
2
0
existed in human, animals, and plants, are cationic in charge.
Many synthetic cationic polymers, such as those based on
a
School of Chemical Engineering, Sichuan University, Chengdu 610065, China
National Engineering Research Center for Biomaterials, Sichuan University,
b
21, 22
23, 24
25, 26
chitosan
, poly(meth)acrylates
, and polycarbonates
,
Chengdu 610064, China
2
7-29
and nylon-3 polymers30, 31, etc.,
and cationic polypeptides
*
Corresponding authors, E-mail: lisai@scu.edu.cn; yjpu@scu.edu.cn
†
Electronic Supplementary Information (ESI) available: molecular property of exhibited excellent, sometimes broad spectrum, antimicrobial
mPEG-PC, DLS results and stability of nanoparticles, in vitro anticancer study and
H&E images of main organs. See DOI: 10.1039/x0xx00000x
activity. Meanwhile, polycationic polymers were widely used as
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a virus mimicking carrier to condense and deliver negatively-
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DOI: 10.1039/C9TB02400J
charged, therapeutic genes to targeted (cancer) cells for gene
therapy.3
2, 33
Similarly, Kataoka et al developed polyion complex
micelles (PIC), composed of cationic and anionic polymers, for
efficiently loading and delivery of charged proteins and nucleic
acids.3
4-36
For example, they used poly(ethylene glycol)-b-
poly(aspartic acid) and a porphyrin-cored third generation
dendrimer decorated with 32 primary amine groups to prepare
3
7
PS-loaded PICs for PDT of lung cancer.
Herein, we reported a cationic polymer-based DDS for
efficient co-loading of oppositely-charged drugs; cationic
anticancer drug (DOX) and anionic PS (Ce6) were employed as
model drugs to demonstrate the feasibility of this strategy. A
block copolymer of methoxy polyethylene glycol (mPEG) and
polycarbonates (PCs) (mPEG-PC, Scheme 2) was designed for
Scheme 2 The synthetic illustration of polycationic block
copolymer mPEG-PC.
the outstanding biocompatibility and biodegradability of PEG
Experimental
Synthesis of mPEG-PC
and PC.1
7, 38-40
PC block was functionalized with primary amine
groups to render the polymer cationic charges. DOX and Ce6
were efficiently loaded into the polymeric nanoparticles by
multiple intermolecular interactions between polymer and
payloads (Scheme 1). The impact of polymer-payload
interactions on the drug loading efficiency, nanoparticle
stability, and drug release behaviors were studied. The in vitro
and in vivo combinational chemo-photodynamic therapy of
murine breast cancer were further investigated to establish the
anticancer efficiency of this DDS.
The synthetic scheme of mPEG-PC is shown in Scheme 2.
Synthesis of 5-methyl-5-allyloxycarbonyl-propylene carbonate
(
MAC). The synthesis of MAC was according the previous work
41
reported by Jing et al. Firstly, 2,2-bis(hydroxymethyl)propionic
acid (18.0 g, 134 mmol) and KOH (8.6 g, 153 mmol) were
dissolved in 150 mL of DMF at 100 ⁰C. Allyl bromide (11.6 mL,
1
4
34 mmol) was added dropwise and the mixture was stirred for
8 h at 45 ⁰C. DMF was removed under vacuum and the residue
was dissolved in 200 mL dichloromethane and extracted by
deionized water (100 mL × 2). The organic phase was combined
and dried with anhydrous Na
dichloromethane under vacuum,
bis(hydroxymethyl)propionate was obtained with a yield of
6%. Secondly, allyl 2,2-bis(hydroxymethyl)propionate (14 g, 80
mmol) and ClCO Et (22.86 mL, 240 mmol) were dissolved in 200
mL anhydrous tetrahydrofuran (THF). Triethylamine (33 mL,
.24 mol) was added dropwise into the stirring mixture at 0 ⁰C
2
SO
4
.
After removal of
allyl 2,2-
5
2
0
within 30 min. The mixture was then stirred at room
temperature for 2 h. The precipitate was filtered off and THF
was removed to obtain crude products, which were
recrystallized from THF/ethyl ether for three times to give the
white crystals MAC (yield：62%).
Synthesis of mPEG-PMAC. mPEG-PMAC was prepared via ring-
opening polymerization of MAC using mPEG2000 as a macro-
initiator. Briefly, mPEG2000 (0.3 g, 0.15 mmol) was added into the
polymerization tube and stirred at 100 ⁰C under vacuum for 2 h
2
to remove trace water. MAC and Sn(Oct) in dry toluene were
added quickly into the cooled tube. After purged with nitrogen
gas for three times, the tube was sealed and immersed in an oil
bath at 110 ⁰C for 24 h. The mixture was dissolved in DCM and
precipitated into excessive cold diethyl ether to obtain mPEG-
PMAC.
Synthesis of mPEG-PC. mPEG-PMAC (0.5 g, 1.14 mmol),
cysteamine hydrochloride (772 mg, 6.80 mmol), and DMPA
Scheme 1 Schematic illustration of utilization of cationic
polymer, mPEG-PC, for differential loading of DOX and Ce6 and
the combinational chemo/photodynamic therapy.
(69.7 mg, 0.272 mmol) were dissolved in 25 mL DMF under
argon atmosphere. The mixture was stirred at room
temperature under UV irradiation (365 nm) for 3 h. Afterwards,
the mixture was dialyzed against deionized water in a dialysis
2
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bag (MWCO 2000) for 2 d. The freeze-dried product was The cytotoxicity of mPEG-PC and DNPs against NIHVi/e3wTA3rticalenOdn/lioner
dissolved in methanol and then precipitated in diethyl ether. 4T1 cells was measured by an MTT assayD. OCIe: l1l0s.1w03e9r/eCs9eTBe0d2e4d00inJ
3
The precipitate was then dried in vacuum to obtain mPEG-PC.
96-well plates (Corning Inc., New York) at a density of 8 × 10
3
NIH/3T3 cells/well or 4 × 10 4T1 cells/well and cultured for 24
Preparation and characterization of drug-loaded mPEG-PC
nanoparticles
h. Culture medium containing different concentrations of
mPEG-PC or DNPs was then added. After 12 h，for the mPEG-
PC cytotoxicity study, cells in the irradiation group were
illuminated with a 660 nm light for 10 min. For the DNPs
cytotoxicity study, cells in the irradiation groups were
illuminated for 0.5, 1, and 3 min. Cells were then incubated for
another 12 h. Thereafter, the medium was discarded and
replaced with 100 µL of MTT in serum-free medium (0.5
mg/mL). After incubation for 4 h, medium was removed and 100
µL DMSO was added into each well. The absorbance at 490 nm
was measured by a microplate reader. The cell viability was
expressed as (sample/control) × 100%. All data were presented
as mean ± SD (n = 3).
The drug-loaded mPEG-PC nanoparticles were prepared by
nanoprecipitation. Different rations of Ce6 and DOX and mPEG-
PC (10 mg) were dissolved in DMSO (1 mL), and the solution was
ultrasonicated for 10 min. Thereafter, the solution was added
dropwise into stirring deionized water (10 mL) and stirred
overnight in the dark. After dialysis against deionized water in a
dialysis bag (MWCO 1000) for 24 h, the mixture was centrifuged
(3000 rpm, 5 min) to remove unloaded DOX and Ce6. The
supernatant was freeze-dried to obtain mPEG-PC-Ce6-DOX
nanoparticles (DNPs). The drug loading contents (DLCs) and
drug loading efficiencies (DLEs) of Ce6 and DOX were
determined by fluorescence spectroscopy at 687 and 585 nm
4
2
Cellular uptake
(Hitachi F-7000), respectively.
The blank mPEG-PC
Cellular uptake in the dark. For cytometry, 4T1 cells were
seeded in 6-well plates (1 × 10 cells/well) and cultured at 37 ⁰C
nanoparticles were prepared similarly.
5
Stability of DNPs
for 24 h. Cells were then treated with culture medium
containing DNPs (DOX: 3 μg/mL) for 1, 2, and 4 h. After
trypsinization and centrifugation (2500 rpm, 3 min), cells were
rinsed with PBS twice and re-suspended in 350 μL PBS for study
on a FACScan instrument (Becton Dickinson, Accuri C6). The
DNPs solution containing 10% FBS was incubated in a shaking
bed at 37 ⁰C with a shaking rate of 150 rpm. The size changes of
DNPs were monitored by DLS.
pH titration of mPEG-PC
The pH value of mPEG-PC solution (2 mg/mL) was adjusted to DOX fluorescence was detected with excitation wavelength at
0 by aqueous NaOH (0.2 mol/L). Aqueous HCl (0.01 mol/L) was 488 nm and emission wavelength at 585 nm. For CLSM study,
1
added dropwise until the pH of mPEG-PC aqueous solution 4T1 cells were seeded on 35 mm diameter glass dishes at a
4
declined to 4.0.
In vitro drug release
density of 2 × 10 cells/mL and cultured for 24 h. Cells were then
treated with medium containing DNPs for 1, 2, and 4 h. Cells
were stained with Hoechst for 20 min, washed, and immersed
in PBS for CLSM observation. DOX and Hoechst were excited at
The release kinetic profiles of DOX and Ce6 from DNPs were
measured by a dialysis method. Drug-loaded nanoparticles
were dispersed in phosphate buffer solutions with different pH
values (pH 7.4 and 5.0, 10 mM). The solutions were transferred
into dialysis bags (MWCO 2000), which were immersed in 15 mL
of buffer solution (containing 0.2% Tween 80) of different pH
values in dark and incubated in a shaking bed at 37 ⁰C with a
shaking rate of 150 rpm. At predetermined time intervals, 1 mL
of medium with released drug was taken out for measurement
and 1 mL fresh medium was supplemented. The released Ce6
and DOX were respectively detected at 687 and 585 nm on a
fluorescence spectrophotometer. For the drug release with the
light irradiation, the protocol was similar except that the DNPs
in dialysis bags were irradiated (660 nm, 3 min) at 4, 8, and 12
4
88 and 346 nm with emission at 585 and 460 nm, respectively.
Cellular uptake with light irradiation. 4T1 cells were seeded in
-well plates (1 × 10 cells/well) and cultured for 24 h. Cells were
then treated with culture medium containing DNPs (Ce6: 5
μg/mL) with or without light irradiation for 3 min and then
cultured for 2 h. The following protocol was similar to that of
the cellular uptake in the dark.
Intracellular drug release
T1 cells were seeded on 35 mm diameter glass dishes and
cultured for 24 h. Cells were then treated with DNPs containing
medium (DOX: 3 μg/mL) for 0.5 h. The culture medium was
replaced by fresh medium and cells were further incubated for
h. Dishes were rinsed with PBS for three times and immersed
in 1 mL PBS for CLSM study.
5
6
4
5
h. The released amount of Ce6 was measured similarly.
1
Singlet oxygen ( O
2
) detection by SOSG
Intracellular ROS detection
DNPs were dispersed in PBS with different pH values to a
concentration of 0.02 mg/mL. After a 6-h incubation in a shaking
bed (150 rpm) at 37 ⁰C. DNPs suspension (100 µL) was diluted
to 1 mL by DI water. SOSG solution in DI water (containing 0.3%
methanol, 50 µL) was added into the diluted DNPs suspension
and the working concentration of SOSG was 1.5 µM. The
fluorescence of the suspension at 525 nm was measured after
light irradiation (λ = 660 nm) of 3, 8 or 13 min.
4
T1 cells were seeded on 35 mm diameter glass dishes at a cell
4
density of 2 × 10 cells/mL and cultured for 24 h. Cells were then
treated with DNPs containing medium (DOX: 3 μg/mL) for 2 h.
Culture medium was replaced by serum-free medium
containing DCFH-DA (10 μM). After 20 min, cells were treated
with or without light irradiation for 3 min. Dishes were rinsed
with PBS for three times and immersed in 1 mL PBS for CLSM
study. DCF was excited at 488 with emission at 525 nm.
Cytotoxicity test
5
For cytometry, 4T1 cells were seeded in 6-well plates (1 × 10
cells/well) and cultured for 24 h. Cells were then treated with
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culture medium containing DNPs (DOX: 3 μg/mL) for 2 h. Culture
medium was replaced by serum-free medium containing DCFH-
DA (10 μM). After 20 min, cells were harvested and rinsed with
PBS twice. Samples were finally re-suspended in 350 μL PBS and
then irradiated for 0, 3, and 5 min. Cells treated with blank
culture medium was used as a control. The fluorescence
intensity of DCF in 4T1 cells was detected with excitation
wavelength at 488 nm.
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DOI: 10.1039/C9TB02400J
Subcellular localization
4
T1 cells were seeded on 35 mm diameter glass dishes at a cell
4
density of 2 × 10 cells/mL and cultured for 24 h. Cells were then
incubated with DNPs containing medium (DOX: 3 μg/mL) for 2
h. The medium was replaced with fresh medium, and cells were
then treated with or without irradiation for 30 s and then
cultured for additional 1 h. Cells were stained with lysotracker
for 30 min, rinsed with PBS for three times, and immersed in 1
mL PBS for CLSM test.
1
6
Fig. 1 H NMR spectrum of mPEG-PC in DMSO-d . (* shows the
residual peak of unmodified polymer side chains and #
represents the residual peak of diethyl ether.)
Ex vivo biodistribution
Male Balb/c mice (6 weeks) were purchased from Dashuo
Biotechnology Co. Ltd. (Chengdu, Sichuan). 4T1 cells (1 × 10 )
were injected into mice right flanks subcutaneously. Ce6+
DOX·HCl (Ce6 of 5 mg/kg; DOX·HCl of 3 mg/kg) and DNPs
Scheme 1 outlines the strategy of preparation of Ce6 and DOX
co-loaded nanoparticles by supramolecular assembly of mPEG-
PC, Ce6, and DOX. Structurally, photosensitizer Ce6 and
chemotherapeutics DOX have hydrophobic and aromatic
groups; besides, they have negative carboxylic groups and
cationic amine groups, respectively. A polycationic block
copolymer mPEG-block-polycarbonate, mPEG-PC, was used to
co-load DOX and Ce6. The hydrophobic and cationic
polycarbonate segments render strong intermolecular
interactions between polymers and cargos including
hydrophobic interactions, hydrogen bonding and electrostatic
interactions (Scheme 1). Meanwhile, strong interactions
between Ce6 and DOX, including electrostatic, π−π stacking and
hydrophobic interactions, also favor the nanoparticle
6
(equivalent Ce6 of 5 mg/kg; equivalent DOX of 3 mg/kg) were
intravenously injected via tail vein when tumor volume reached
3
about 200 mm . At predetermined times, mice were sacrificed,
and the main organs and tumors were collected and imaged ex
vivo by an in-vivo imaging system (Maestro).
In vivo antitumor study
The in vivo studies were carried out in compliance with the
Animal Management Rules of the Ministry of Health of the
People’s Republic of China (Document no. 55, 2001) and the
institutional guidelines. The experiments were approved by the
Animal Care and Use Committee of Sichuan University. 4T1
tumor-bearing mice were randomly divided into four groups (n
1
9
formation.
The synthesis of mPEG-PC was illustrated in Scheme 2. Firstly,
cyclic carbonate monomer MAC containing carbon-carbon
double bond was synthesized via successive esterification and
cyclization of 2,2-bis(hydroxymethyl)propionic acid. The
3
=
5) when tumor volume reached 100-150 mm . Four kinds of
formulations were administrated via tail intravenous injection
on days 0, 3, and 6: (1) Saline; (2) Ce6 (5 mg/kg); (3) DOX·HCl (3
mg/kg); and (4) DNPs (equivalent Ce6 of 5 mg/kg; equivalent
DOX of 3 mg/kg). For Ce6 and DNPs groups, tumor was
1
chemical structure of MAC was verified by H NMR (Fig. S1).
mPEG-PMAC was then synthesized by ring opening
2
polymerization of MAC using mPEG as macro-initiator and
irradiated (660 nm, 100 mW/cm , 10 min) 6 h post-injection.
1
Sn(Oct)
2
as catalyst. The H NMR spectrum of mPEG-PMAC was
The tumor volumes and body weights of mice were measured
every 2 days. After 12 days, mice were sacrificed. The main
organs (heart, liver, spleen, lung, and kidney) and tumors were
excised to fix in 4% paraformaldehyde and then stained for H&E
studies.
Statistical analysis
Student t-test was used to conduct statistical analysis. Statistical
significance was set at *p < 0.05, ** p < 0.01, *** p < 0.001.
shown in Fig. S2; peaks f and b were ascribed to the allyl and
methene protons in the polycarbonate and mPEG segments.
The degree of polymerization was calculated to be 12 and the
molecular weight of mPEG-PC was 4400 g/mol by NMR. Gel
permeation chromatography (GPC) was studied to further
confirm the successful polymerization; the number-average
molecular weight of mPEG-PMAC was measured to be 8000
g/mol (Fig. S3 and Table S1).
mPEG-PC was then synthesized by thiol-ene click reactions
between the thiols in cysteamine hydrochloride and the carbon-
Results and discussion
Synthesis and characterization of mPEG-PC
4
3
carbon double bonds in mPEG-PMAC (Scheme 2). The
1
successful synthesis of mPEG-PC was demonstrated by H NMR
(Fig. 1). The new peaks i (2.95−2.98 ppm) and h (2.68−2.72 ppm)
corresponding to the methene protons of the pristine
cysteamine appeared, suggesting successful chemical
modification. The intensity of peaks at 5.81−5.91 ppm and
4
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5
.17−5.31 ppm that were ascribed to the allyl protons 5). The formation of nanoparticles was confirmed by tVhieew cAortiuclnetOrnalitnee
decreased sharply. The percentage of modification was results, which were obtained by dynamic ligDhOtI:s1c0a.t1t0e3r9in/Cg9(TDBL0S2)4a0n0dJ
calculated to be 85% by comparison of these peaks’ areas. summarized in Table 1. The blank polymeric NPs showed very low
Primary, secondary, and tertiary amine containing polymers count rate (13 kcps), indicating its poor ability to generate
can be protonated at acidic pH, for example in acidic organelles nanoparticles, which was probably due to the low hydrophobicity of
(endosomes and/or lysosomes), showing a pH-responsive mPEG-PC. In contrast, DOX- and/or Ce6-loaded NPs showed much
property. To study the pH-responsiveness of mPEG-PC, we higher count rates (> 10 times), suggesting the successful
4
4
conducted an acid-base titration of mPEG-PC solution. The nanoparticle formation. The multiple interactions including
pH-sensitive range of mPEG-PC was determined to be pH 8.4 to electrostatic interactions, hydrophobic, and π-π interactions
4
.9 by the first order derivation of the titration curve (Fig. S4).
between drugs and mPEG-PC collectively facilitated the nanoparticle
preparation (Scheme 1). The NPs showed a diameter of tens of
nanometers by DLS and positive zeta potentials in water. (Fig. S5 and
Table 1).
Preparation and characterization of blank and drug-loaded
polymeric nanoparticles
We then used mPEG-PC to prepare blank and drug-loaded NPs by
adjusting the feeding weight ratios of Ce6 to DOX (Table 1, NP-1 to
Table 1 DLCs, DLEs, sizes, and zeta potentials of drug-loaded polymer nanoparticles.
a
a
Zeta
potential
Size
nm)
Count rate
(kcps)
Feeding weight ratio of
Ce6 and DOX
DLCs
(%)
DLEs
(%)
b
Samples
(
(mV)
Blank
NP-1
-
-
-
103.1
+22.4
13
Ce6
DOX
Ce6
DOX
Ce6
DOX
Ce6
DOX
Ce6
DOX
0%
50%
15%
35%
25%
25%
35%
15%
50%
0%
-
-
35.12
25.65
+26.7
+19.8
+6.79
+5.89
+4.08
148
348
287
256
243
14.0
7.0
11.9
13.8
9.4
15.4
5.3
16.4
-
28.0
46.7
34.0
55.2
37.6
44.0
35.3
32.8
-
NP-2
NP-3
33.90
(
DNPs)
NP-4
50.06
39.56
NP-5
a. The concentration of NPs was 0.4 mg/mL.
b. Tested in deionized water.
The NPs with higher loading capacity of DOX showed higher zeta confirmed by the zeta potential results (Table 1); the higher DLC of
potential owing to the positive charge nature of DOX. The successful negatively-charged Ce6, the lower zeta potential of NPs in water.
loading of DOX and Ce6 was verified by UV absorption. As shown in Furthermore, the hydrophobic interactions in DNPs were
1
50
Fig. 2A, the characteristic peaks of DOX and Ce6 at 480 and 411 nm demonstrated by H NMR. As shown in Fig. S7, the peaks of DOX
1
appeared in the UV absorption of DNPs in DMSO. A red shift of these and Ce6 were not found in the H NMR spectrum when D
2
O was used
two peaks in the UV spectrum of DNPs in water was ascribed to the as a solvent. In stark contrast, peaks of Ce6 and DOX at low field
π-π interactions in the DNPs.45, 46 The DOX and/or Ce6 loading appeared when DMSO-d
, a good solvent of polymer and drugs, was
efficiency and capacity by mPEG-PC were then studied by used. These NMR results verified that hydrophobic interactions were
6
1
9, 47-49
fluorescence spectroscopy (Fig. 2B).
Free DOX and Ce6 emit present in DNPs and DOX and Ce6 were mainly dispersed in the
strong fluorescence at 585 and 670 nm when excited at 480 and 400 hydrophobic core.
nm, respectively (Fig. S6). Because dimethyl sulfoxide (DMSO) is a
good solvent for both polymer and drugs, DMSO was used as solvent
to disintegrate NPs and adequately dissolve free drugs for
fluorescence study. As shown in Table 1, when the total feeding
amount of DOX and Ce6 was fixed (50wt% in polymer + drug system),
the DLCs would increase as the increase of the feeding ratio of drugs.
NP-3 showed the highest total DLCs of 23.2% (13.8% and 9.4% for
Ce6 and DOX, respectively). It should be noted that in all groups the
DLEs of Ce6 were higher than those of DOX in the co-loaded NPs (NP-
2
, 3, and 4). The higher DLE of Ce6 was ascribed to the strong
electrostatic attraction between negatively-charged Ce6 and
positively-charged mPEG-PC. The electrostatic interactions were also
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DOI: 10.1039/C9TB02400J
Fig. 3 In vitro drug release of DOX and Ce6 from DNPs in
different pH conditions.
Fig. 2 UV absorption (A) and fluorescence spectroscopy (B, in
DMSO) of DNPs. TEM images of DNPs in neutral (C) and acidic
To further understand the underlying mechanism of
h. differential drug release, we further studied the size and
morphology of DNPs in acidic condition. As shown in Figure 2C
and D, the DNPs in acidic condition showed comparable size
with and similar morphology to that in neutral condition,
suggesting that the nanoparticles were not rapidly
disassociated by protonation of mPEG-PC. Thereafter, the zeta
potentials of DNPs in different pH conditions were studied (Fig.
S9). DNPs showed negative (−1.77 mV) and positive (+1.45 mV)
zeta potentials in pH 8.4 and 5.0, respectively, indicating the
(D)
environment
for
4
The stability of these NPs was studied by dilution (Fig. S5). DOX-
loaded NP-1 and DOX and Ce6 co-loaded NP-2 showed a size change
after dilution, suggesting a relatively weak stability. However, other
co-loaded NPs (NP-3 and NP-4) and Ce6-loaded NPs (NP-5) showed
better stability against dilution and no obvious size changes were
observed after dilution, suggesting their more intense intermolecular
interactions in these NPs. Considering the highest DLEs and that
protonation of mPEG-PC in acidic condition. Therefore, the acid-
5
1
relatively low zeta potential is conductive to long blood circulation , accelerated release of DOX could be partly ascribed to the
we utilized the co-loaded NP-3 (denoted as DNPs) for the following electrostatic repulsion of positively-charged, protonated DOX
4
5, 52
study. The TEM image (Fig. 2C) manifested that DNPs were spherical and polymers.
Given the low cationic charge and relatively
stable nanoarchitecture of DNPs at pH 5, a rational explanation
could be that mPEG-PC was partially protonated in acidic
condition but the nanoparticles could be still stabilized by the
intense interactions between mPEG-PC and Ce6.
The generation of singlet oxygen by DNPs in aqueous was
then studied by using singlet oxygen sensor green (SOSG) as a
fluorescence probe. DNPs were suspended in PBS (pH 7.4 and
with a diameter of about 30 nm, comparable with the DLS size (Table
). The stability of DNPs in 10% FBS was also studied (Fig. S8).
Negligible size changes were observed after a 24-h incubation,
implying that DNPs could possess favorable stability in circulation.
1
In vitro drug release and singlet oxygen generation under light
irradiation
5
.0) for 6 h and then irradiated by laser light of 660 nm for
The in vitro drug release behavior of DNPs was investigated in
different pH conditions (PBS, pH 7.4 and 5.0. Fig. 3). DNPs showed
comparable and slow release of Ce6 (< 40% at 48 h) in pH 7.4 and pH
different irradiation times. As shown in Fig. S10, DNPs in pH 5.0
generated more singlet oxygen than those in pH 7.4, which was
probably due to easy diffusion of the generated singlet oxygen
5
.0. However, the DOX release from DNPs was pH-responsive. The
53
after the rapid release of DOX in acidic condition.
DNPs showed a sustained and minimal drug release in neutral pH
condition; about 40% DOX was released within 48 h. However, a
much faster release of DOX was observed at pH 5.0 and about 80%
was released at 48 h. The different release behaviors of DOX and Ce6
from DNPs in different pH conditions were ascribed to their different
solubility in acidic conditions and their distinct interactions with
In vitro anticancer activity
The cytocompatibility of mPEG-PC was first studied before the
in vitro anticancer study of DNPs. The cytotoxicity of mPEG-PC
against NIH/3T3 and 4T1 cells were studied by an MTT assay. As
shown in Fig. S11, mPEG-PC showed low cytotoxicity and the
viability of NIH/3T3 cells was more than 80% even at a high
polymer carrier. In acidic condition, primary amine of DOX is polymer concentration of 50 μg/mL. In addition, similar results
protonated and the water solubility could be significantly improved, were obtained when cells were treated with mPEG-PC and light
resulting into accelerated diffusion and drug release. In contrast, Ce6 irradiation, suggesting that mPEG-PC had no phototoxicity.
showed slight slower release in acidic condition owing to the reduced
solubility of carboxyl groups. Compared with the carrier-free
1
9
nanodrugs, DNPs showed much slower in vitro drug release, which
was probably due to the protection of PEG shell and intense polymer-
payloads interactions and conductive to the less drug leakage in
blood circulation.
6
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DOI: 10.1039/C9TB02400J
Fig. 5 Flow cytometry (A, B) results of 4T1 cells treated with
DNPs for different incubation times in the absence of
irradiation.(C) CLSM images and (D) nucleus co-localization
analysis of 4T1 cells treated with DNPs for 1, 2, and 4 h.
Fig. 4 The cytotoxicity of Ce6, DOX·HCl, the physical mixture of Light-triggered enhanced cellular uptake and endosomal escape
Ce6 and DOX·HCl (Ce6+ DOX·HCl), and DNPs against 4T1 cells
after a 24 h incubation without (A) and with (B) light irradiation
The cellular uptake behaviors of DNPs by 4T1 cells in the dark
were first studied by flow cytometry. 4T1 cells were treated
of 660 nm for 30 s. (n = 3, ** p < 0.01, *** p < 0.001.). with DNPs for 1, 2, and 4 h. The DNPs showed a time-dependent
internalization behavior and the more cellular uptake of DNPs
The in vitro anticancer activity of DNPs in 4T1 cells with and were observed with longer incubation time (Fig. 5A and B).
without light irradiation were then studied; DOX·HCl, Ce6 and Similar results were observed in the CLSM results (Fig. 5C), and
their physical mixture (Ce6+DOX·HCl) were used as control the intracellular co-location analysis (Fig. 5D) showed that some
groups. As shown in Fig. 4, cells treated with DOX·HCl showed DOX had diffused into nucleus, where DOX can exert the
negligible cytotoxicity differences with and without light anticancer effect by insertion into DNA. Since polymer
irradiation because DOX·HCl is not a photosensitizer. In nanoparticles are generally internalized by cancer cells in a
contrast, Ce6 showed the least dark cytotoxicity yet significantly manner of endocytosis, the acidic environment in
enhanced anticancer efficacy under light irradiation. For endo/lysosomes could trigger the intracellular DOX release of
example, the viability of 4T1 cells treated with 5 μg/mL Ce6 with DNPs. As shown in Fig. S13, the co-localization results of DOX
and without light irradiation was 32.4% and 83.0%, respectively. and Ce6 indicated that distribution of DOX and Ce6 were not co-
Ce6+DOX·HCl and DNPs showed comparable anticancer activity localized in the same region, suggesting the intracellular DOX
5
4
in the dark. When under irradiation, DNPs and Ce6+DOX·HCl release from DNPs.
showed better anticancer activity than the other two groups,
We then studied the cellular uptake of DNPs by 4T1 cancer
suggesting synergistic anticancer effect of Ce6 and DOX. cells under light irradiation. 4T1 cells were cultured with DNPs
Furthermore, combination index (CI) values of PDT and containing medium and treated with and without light
chemotherapy in DNPs were calculated and summarized in irradiation for 3 min. The cells were then cultured for 2 h to
Table S2. The results (CI < 1) demonstrated that DNPs study the impact of light irradiation on the cellular uptake of
performed excellent synergistic effect on the inhibition of DNPs. As shown in Fig. (6A and B), a significant increase of
cancer cell proliferation.
intracellular mean fluorescence intensity of Ce6 was observed
The impact of irradiation time on the anticancer efficacy of after light treatment. To explore whether the irradiation
DNPs was also studied (Fig. S12). The prolonged irradiation time accelerated the extracellular release of Ce6 and induced more
led to enhanced cancer cell inhibition. The IC50s (Table S3) of cellular uptake of Ce6, the in vitro release of Ce6 under
DNPs were 9.22, 6.60, and 5.58 μg/mL when the irradiation time irradiation was studied (Fig. S14). Obviously, the light irradiation
was 0.5, 1, and 3 min, respectively. The longer irradiation time showed negligible effect on the release of Ce6. Therefore, the
could generate more ROS in cancer cells, resulting in more cell enhanced cellular uptake of DNPs after light irradiation was
damages and better PDT outcomes.
ascribed to the Ce6-mediated generation of ROS that induced
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lipid peroxidation and thereby improved cell membrane DOX need to escape from these organelles, diffuse into nucleus,
View Article Online
permeability, which was also known as photochemical
DOI: 10.1039/C9TB02400J
and insert into DNA to exert its anticancer activity. Because ROS
internalization (PCI).5
5, 56
could destroy bio-membranes, we then tested whether the
organelle membranes could be destroyed after light irradiation
Fig. 6E and F). Obviously, the red fluorescence of DOX and the
To verify the ROS generation in PDT, intracellular ROS of 4T1
cells treated with DNPs in the presence and absence of light
irradiation was measured by DCFH-DA. DCFH-DA per se has no
fluorescence and can pass through cell membrane freely. After
internalization, DCFH-DA can be hydrolyzed by esterase to give
(
green fluorescence of lysotracker overlapped to a large extent
when cells were treated without irradiation, suggesting that
DNPs were mainly in the lysosomes and DNPs were internalized
by endocytosis. In contrast, after light irradiation for 0.5 min,
the green fluorescence was hardly observed, suggesting that
lysosomal membranes were destroyed by ROS generated in
PDT. The light-triggered ROS-induced lysosomal membrane
damage could boost the endo/lysosomal escape of DNPs and
release of DOX, realizing synergistic cancer therapy.
DCFH that could be oxidized by ROS to give DCF with green
fluorescence.5
7, 58
The cells were treated with DNPs for 2 h and
then irradiated for 3 min. The light irradiation group (+L)
showed stronger green fluorescence intensity, confirming the
ROS generation after light irradiation (Fig. 6C). It could be
observed there was weak DCF fluorescence in the DNPs group
without irradiation treatment, which may be ascribed to the
Biodistribution and in vivo antitumor efficacy of DNPs
5
9, 60
endogenous ROS and induction from DOX·HCl.
Meanwhile,
Encouraged by the in vitro synergistic anticancer effect, the in
vivo biodistribution of DNPs was investigated by ex vivo
fluorescence imaging. 4T1 tumor-bearing mice were divided
into two groups and intravenously injected with DNPs and the
physical mixture of Ce6 and DOX·HCl (Ce6+DOX·HCl). The main
organs and tumors of mice were collected at desired time points
6 and 24 h) and the fluorescence images were show in Fig. 7A.
Tumor in DNPs group showed significantly enhanced
fluorescence intensity relative to that of Ce6+DOX·HCl group at
flow cytometry was employed for a quantitative analysis of the
generated ROS (Fig. 6D). The irradiated group presented higher
mean fluorescence intensity of DCF, which was consistent with
the CLSM result.
Nanoparticles are generally internalized by cancer cells in a
manner of endocytosis, after which the nanoparticles could be
(
6
1
trapped into acidic endosomes and/or lysosomes. DNPs could
accelerate the DOX release in the acidic conditions; however,
6
h, suggesting the improved accumulation of DNPs by
6
2
enhanced permeation and retention (EPR) effect. Meanwhile,
strong DOX fluorescence was also observed in the kidneys in the
DNPs group at 6 h, implying that DNPs were probably cleared
by
kidney.
Fig. 6 Flow cytometry analysis (A, B) of DNPs-treated 4T1 cells
at 2 h with (+L) and without (-L) the pretreatment of light
irradiation for 3 min. (C) CLSM images and (D) flow cytometry
analysis for ROS generation of 4T1 cells treated with DNPs with
(
+L) and without light irradiation (-L). (E) CLSM images and (F)
Fig. 7 Ex vivo FL images (A) and average signal (B) of main organs and
tumors after intravenous injection of DNPs and DOX·HCl.
lysosomal co-localization analysis of 4T1 cells incubated with
DNPs with (+L) and without light irradiation (-L).
8
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Journal Name
ARTICLE
The in vivo antitumor efficacy of DNPs was explored on 4T1 tumor- release, and efficient endo/lysosome escape of nanoparticles
View Article Online
3
DOI: 10.1039/C9TB02400J
bearing mice. When tumor volume reached 100-150 mm , different mediated by irradiation-induced ROS. In vivo study showed that
formulations (Saline, DOX·HCl, Ce6, and DNPs) were administrated nanoparticles-based chemo-photodynamic combination
via intravenous injection. The tumors of mice in groups of Ce6 and therapy had better antitumor efficacy than monotherapy. Our
DNPs were further irradiated by 660 nm laser. As shown in Fig. 8, study provided a strategy of using polycationic polymer for
DOX·HCl group showed poor tumor inhibition because of the low differential encapsulation and release of oppositely-charged
6
3, 64
DOX dosage (3 mg/kg) than normal one (5 mg/kg).
The Ce6 payloads, which could have great potential in various
group efficiently suppressed the tumor growth in the first 8 days; combination therapies.
however, the tumor volumes rapidly increased one PDT was ceased
on day 6. DNPs showed a significantly enhanced tumor inhibition
relative to these two groups, indicating that the combinational
Conflicts of interest
chemo-photodynamic therapy was superior to monotherapy. The
relative tumor volumes of Ce6, DOX·HCl, and DNPs groups on day 12
were 6.64, 6.95, and 4.51 times of the corresponding average tumor
There are no conflicts to declare.
volumes on day 0. Meanwhile, the body weights of mice were Acknowledgements
monitored for preliminary safety evaluation. There were no obvious
The authors thank for the financial support of National Natural
Science Foundation of China (No. 51903172, 51773130 and
1573111), Sichuan Science and Technology Program
2019YJ0051), and Fundamental Research Funds for the Central
Universities (YJ201764). We would like to thank the Analytical
Testing Center of Sichuan University for NMR and TEM tests.
body weight changes in all groups during the short-term treatment.
Hematoxylin and eosin (H&E) assays were further carried out to
confirm the antitumor efficacy and organ toxicity. As presented in
Fig. S15, DNPs showed negligence damage to the normal organs. No
obvious body loss and cardiomyopathy was observed in the DOX·HCl
group, which was probably due to the low DOX dose of 3 mg/kg.
5
(
&
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Journal of Materials Chemistry B
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DOI: 10.1039/C9TB02400J
We reported a strategy of using polycationic polymer to realize distinct loading and
release of oppositely-charged payloads and a combination therapy.