Fabrication of Reductive-Responsive Prodrug Nanoparticles with Superior Structural Stability by Polymerization-Induced Self-Assembly and Functional Nanoscopic Platform for Drug Delivery

Article abstract of DOI:10.1021/acs.biomac.6b00819

A highly efficient strategy, polymerization-induced self-assembly (PISA) for fabrication of the polymeric drug delivery systems in cancer chemotherapy is reported. Diblock prodrug copolymer, PEG-b-P(MEO₂MA-co-CPTM) was used as the macro-RAFT ag

Full text of DOI:10.1021/acs.biomac.6b00819

Article
pubs.acs.org/Biomac
Fabrication of Reductive-Responsive Prodrug Nanoparticles with
Superior Structural Stability by Polymerization-Induced Self-
Assembly and Functional Nanoscopic Platform for Drug Delivery
Wen-Jian Zhang, Chun-Yan Hong,* and Cai-Yuan Pan*
CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and
Technology of China, Hefei, Anhui 230026, China
S
* Supporting Information
ABSTRACT: A highly efficient strategy, polymerization-
induced self-assembly (PISA) for fabrication of the polymeric
drug delivery systems in cancer chemotherapy is reported.
Diblock prodrug copolymer, PEG-b-P(MEO2MA-co-CPTM)
was used as the macro-RAFT agent to fabricate prodrug
nanoparticles through PISA. The advantages of fabricating
intelligent drug delivery system via this approach are as
following: (1) Simultaneous fulfillment of polymerization, self-
assembly, and drug encapsulation in one-pot at relatively high
concentration (100 mg/mL); (2) Almost complete monomer
conversion allows direct application of the resultant prodrug
nanoparticles without further purification; (3) Robust structures
of the resultant prodrug nanoparticles, because the cross-linker
was used as the comonomer, resulted in core-cross-linking simultaneously with the formation of the prodrug nanoparticles; (4)
The drug content in the resultant prodrug nanoparticles can be accurately modulated just via adjusting the feed molar ratio of
MEO2MA/CPTM in the synthesis of PEG-b-P(MEO2MA-co-CPTM). The prodrug nanoparticles with similar diameters but
various drug contents were obtained using different prodrug macro-CTA. In consideration of the long-term biological toxicity,
the prodrug nanoparticles with higher drug content exhibit more excellent anticancer efficiency due to that lower dosage of them
are enough for effectively killing HeLa cells.
The structural integrity of the nanocarriers is another
important issue for designing polymeric drug delivery
system.15,16 The polymeric nanocarriers are usually subjected
to high dilution and large shear force during blood circulation.
When the polymer concentration is reduced to below the
critical micelles concentration (CMC), the polymeric nano-
carriers tend to dissociate, which induces undesirable drug
leakage. To enhance stability of the polymeric nanocarriers,
shell cross-linking (SCL) and core cross-linking (CCL)
strategies have been proposed for efficient drug delivery, even
subjecting to severe biological microenvironment.6,17−19
Conventionally, the polymeric drug nanocarriers are
prepared through postpolymerization self-assembly of amphi-
philic block copolymers, during which the anticancer drugs
were encapsulated into the cores of nanocarriers. Such self-
assembly is usually conducted at relatively high dilution (the
polymer concentration is typically less than 1%). In addition,
further purification for removal of the unencapsulated drugs is
usually necessary.20−22 For fabrication of the nanocarriers with
robust structures, additional procedure for core/shell cross-
INTRODUCTION
■
Polymeric drug delivery systems for cancer chemotherapy have
attracted more and more attention during the past decades,
partially due to the limitations of small molecule drugs, such as
severe side effects, limited stability, poor water solubility,
unsatisfactory biodistribution, and pharmacokinetics.1−3 Gen-
erally, amphiphilic block copolymers are used to fabricate
nanocarriers with core−shell nanostructures loading small
chemotherapeutic drugs in the cores. The hydrophilic shell,
such as polyethylene glycol (PEG), can effectively prevent
anticancer drugs in the core from prematurely interacting with
the biological environment, protect the drug from premature
degradation, minimize nonspecific protein adsorption during
blood circulation and capturing by the reticuloendothelial
system (RES). The majority of these nanocarriers demonstrate
passive targeting capabilities due to the characteristic features of
tumor biology that facilitate accumulation of nanocarriers in the
tumor through the enhanced permeability and retention (EPR)
effect.4−8 At designing the nanocarriers, to achieve site-specific
drug release at the pathological site should be always
considered. Since the reductive and mildly acidic intracellular
microenvironment of the tumor cells, lots of pH or reductive
responsive nanocarriers have been reported.9−14
Received: June 4, 2016
Revised: August 2, 2016
© XXXX American Chemical Society
A
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linking is necessary.9,23 In recent years, polymerization-induced
self-assembly (PISA) strategy has become a powerful tool for
the fabrication of polymeric nanoparticles (NPs),24−33 which is
due to the much high fabricating efficiency including very high
fabricating concentration, combination of the polymerization
and the self-assembly in one-pot, almost complete monomer
conversion leading to no further purification for the following
applications. What is more, NPs with a robust structure can be
obtained just by adding cross-linker as comonomer in the
polymerization system without any additional postpolymeriza-
tion procedure.34,35 A few literatures have reported the
encapsulation of guest cargos, such as proteins and nile red,
into the vesicles during the PISA,36−38 but fabricating
nanocarriers with performance of controlled release in response
to biological milieu still remains a great challenge. Davis et al.
fabricated NPs by PISA for drug delivery in 2014,39 but post-
PISA drug conjugation and core-cross-linking should be
conducted. Moreover, styrene was used as the monomer for
PISA, which is incompatible for bioapplication, and further
purification after PISA should be performed to remove the
unreacted styrene due to the low monomer conversion.
Scheme 2. Proposed Mechanism of Reductive-Responsive
CPT Parent Drug Release from the Prodrug NPs
To solve the above problems, we designed superiorly stable
prodrug NPs with three layer structure as shown in Scheme 1.
content is enough for effectively killing HeLa cells, which is
beneficial to reduce the long-term biological toxicity.
Scheme 1. Fabrication of Reductive-Responsive Prodrug
NPs with Robust Structure by Polymerization-Induced Self-
Assembly (PISA) Using PEG-b-P(MEO2MA-co-CPTM) as
the Macro-CTA
EXPERIMENTAL SECTION
■
Material. Methoxypolyethylene glycol amine (PEG113-NH2,
aladdin), N-hydroxysuccinimide (98%, aladdin), camptothecin (98%,
chengdu tianyuan natural product Co., Ltd.), benzyl methacrylate
(BzMA, 98%, energy chemical), cystamine dihydrochloride (99%,
energy chemical), methacryloyl chloride (95%, aladdin), Dulbecco’s
modified Eagle’s medium (DMEM, Hyclone), fetal bovine serum
(FBS, Hyclone) and glutathione reduced form (GSH, ≥98%, Sigma-
Aldrich) were used as received. 2-(2-Methoxyethoxy)ethyl methacry-
late (MEO2MA, Mn ∼ 188 g/mol, 95%) was purchased from Sigma-
Aldrich and purified by passing over a neutral Al2O3 column to remove
the inhibitor. N,N′-Azobis(isobutyronitrile) (AIBN, 98%, aladdin) was
recrystallized from ethanol prior to use. 2-((2-Hydroxyethyl)-
disulfanyl)ethyl methacrylate (HSEMA) and 4-(4-cyanopentanoic
acid) dithiobenzoate (CPDB) were synthesized according to the
literature.40,41 All other reagents of analytical grade were used without
further purification.
Synthesis of 4-Cyano-4-((thiobenzoyl)sulfanyl) Pentanoic
Succinimide Ester (SCPDB). N-Hydroxysuccinimide (2.52 g, 21.9
mmol) and 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid
(CPDB, 6.11 g, 21.9 mmol) were dissolved in anhydrous dichloro-
methane (20 mL). After stirring at ice−water cooling bath for 30 min,
a solution of dicyclohexylcarbodiimide (DCC; 4.53 g, 21.9 mmol) in
anhydrous dichloromethane (10 mL) was added dropwise into the
above mixture over 30 min. The reaction mixture was stirred at 20 °C
in dark for 24 h and then washed with distilled water twice. The
organic layer was dried over anhydrous Na2SO4. After removing the
Na2SO4 by filtration, the solvent was removed using a rotary
evaporator. The crude product was purified by column chromatog-
raphy using a mixed eluent of n-hexane/ethyl acetate = 4:1 (v/v). A
red solid was obtained in 86% yield.
The redox-sensitive core is cross-linked via copolymerization of
benzyl methacrylate (BzMA) and N,N-cystaminebismethacry-
lamide (CBMA); the middle layer is the anticancer drug,
camptothecin (CPT)-containing copolymer, in which CPT is
linked to the polymer chains via reductive-sensitive linkage; and
the outer layer is hydrophilic poly(ethylene glycol) which is
used for stabilize the NPs. Thus, the drug NPs are superiorly
stable even in good solvens, highly soluble in water and
reductive-sensitive. Such prodrug NPs are fabricated through
RAFT dispersion polymerization of (BzMA) and cross-linker,
CBMA using diblock prodrug copolymer PEG-b-P(MEO2MA-
co-CPTM) as the macro-RAFT agent (macro-CTA), so the
prodrug NPs are formed simultaneously with core-cross-linked.
The CPT contents in the macro-CTA can be accurately
modulated just via adjusting the feed molar ratio of CPTM/
MEO2MA, and further the drug content in the resultant
prodrug NPs can be controlled by using different CPT contents
of macro-CTA. Cleavage of the conjugated CPT drug within
the prodrug NPs can be effectively triggered under reductive
microenvironments, such as high level of GSH in the cytosol.
Scheme 2 shows the mechanism of reductive-responsive CPT
parent drug release from the prodrug NPs. Cell viability assays
reveal that the lower dose of the prodrug NPs with higher drug
Synthesis of Poly(ethylene glycol) Dithiobenzoate (PEG113
-
CPDB). All glassware was dried at 100 °C overnight to remove a trace
of water before carrying out the reaction. SCPDB (0.45 g, 1.2 mmol)
was dissolved in anhydrous dichloromethane (10 mL). A solution of
PEG113-NH2 (5 g, 1 mmol) in anhydrous dichloromethane (15 mL)
was added dropwise into the SCPDB solution over 1 h. After stirring at
room temperature for 12 h, the reaction solution was poured into
excess diethyl ether to afford some pink precipitate. The precipitate
was collected by filtration and then dissolved in dichloromethane, and
the precipitation process was repeated for an additional two times. The
degree of dithiobenzoate functionalization was approximately 96% as
1
calculated according to the H NMR data (Figure 1).
B
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In Vitro Redox-Regulated Drug Release. The redox-regulated
drug release was performed as the follows. The four different prodrug
NPs were respectively transferred into the dialysis bags (molecular
weight cutoff: 3500 Da) and dialyzed against 200 mL of PBS buffer
(10 mM, pH 7.4) with glutathione (GSH) at different concentrations
(0.01, 5, and 10 mM) at 37 °C. An aliquot of the buffer solution (2
mL) outside of the dialysis bags was sampled and replaced with equal
volume of fresh buffer solution at the predetermined intervals. The
concentration of the CPT released from the prodrug NPs was
evaluated using the fluorescence spectrometer (excitation wavelength:
340 nm, emission wavelength: 440 nm).
In Vitro Cytotoxicity Assay. The in vitro cytotoxicity was
determined via MTT assay. The cells were first cultured in Dulbecco’s
modified Eagle medium (DMEM) supplemented with streptomycin
(100 μg/mL), penicillin (100 units/mL), and 10% fetal bovine serum
(FBS) at 37 °C in a CO2/air (5:95) incubator for 2 days. For MTT
assay, HeLa cells were seeded in a 96-well plate with an initial density
of 8000 cells/well in 100 μL of complete Dulbecco’s modified Eagle’s
(DMEM) medium supplemented with 10% fetal bovine serum (FBS),
and then cultured in a CO2/air (5:95) incubator at 37 °C for 24 h. The
cells were then treated with the prodrug or drug-free NPs at various
concentrations. Cells without nanomaterials treatment were used as
control. After 48 h of incubation, the media were removed and replace
by fresh DMEM media containing 0.5 mg/mL MTT reagent. After
further 4 h of incubation, the medium in each well was replaced by 150
μL of dimethyl sulfoxide (DMSO) to dissolve the obtained blue
formazan crystals. The absorbance at 570 nm was measured to
evaluate the cell viability.
Cellular Internalization of the Prodrug NPs Observed by
CLSM. HeLa cells were seeded in a cell culture plate with an initial
density of 3 × 104 cells/well in 300 μL of DMEM medium
supplemented with 10% fetal bovine serum (FBS) and then cultured in
a CO2/air (5:95) incubator at 37 °C for 24 h. The media were
removed and replaced by the fresh DMEM medium (supplemented
with 10% FBS) containing prodrug NPs at a final CPT concentration
of 10 μg/mL. After incubation for 4, 8, and 16 h, respectively, the
media were removed and the cells were washed with PBS three times.
The intracellular distribution of drug was observed by a LSM510
confocal laser scanning microscope.
1
Figure 1. H NMR spectrum of PEG113-CPDB in CDCl3.
Synthesis of Reduction-Responsive Monomer (CPTM).
Camptothecin (CPT, 5.0 g, 14.4 mmol) and DMAP (5.26 g, 43.1
mmol) were mixed with anhydrous dichloromethane (300 mL) under
an argon atmosphere. Triphosgene (1.49 g, 5.0 mmol) was added into
the above mixture, and then the mixture was stirred at room
temperature for 30 min. HSEMA (4.01 g, 15.8 mmol) was diluted with
anhydrous dichloromethane (50 mL) and then added dropwise into
the above mixture via a constant pressure funnel. After stirring at room
temperature for 12 h, the mixture was concentrated by a rotary
evaporator. The crude product was purified by silica column
chromatography using ethyl acetate as the running solvent. A pale
solid powder was obtained in 83% yield (6.97 g).
Synthesis of N,N-Cystaminebismethacrylamide (CBMA).
Cystamine dihydrochloride (9.0 g, 40 mmol) was dissolved in 80
mL of water. Aqueous solution of sodium hydroxide (16 mL, 10 M)
was added into the above solution, and then the mixture was stirred in
ice−water bath for 20 min. Methacryloyl chloride (8.36 g, 80 mmol, in
10 mL of dichloromethane) was added dropwise into the above
mixture at 0 °C, while a white precipitate was formed. After
continuously stirring for 3 h, the reaction mixture was filtered, and
the solid was collected and then washed with deionized water for three
times. The product was obtained by crystallization from ethyl acetate
in 47% yield.
Synthesis of Diblock Copolymer PEG-b-P(MEO2MA-co-
CPTM) with Various Contents of CPTM. PEG113-CPDB (0.53 g,
0.1 mmol), AIBN (1.64 mg, 10−2 mmol), and CPTM/MEO2MA, with
a total molar mass of 4.5 mmol and various molar ratios of 0/1, 1/8, 1/
4, and 1/2, were dissolved into 1,4-dioxane (5 mL), and then the
resultant solutions were respectively added into four 10 mL
polymerization tubes. The tubes were sealed under vacuum after
three freeze−evacuate−thaw cycles. Then the polymerizations were
conducted at 70 °C while stirring for 24 h. After rapid cooling to room
temperature, the reaction mediums were added dropwise into the
excess diethyl ether to obtain a precipitate. The precipitation processes
were repeated for three times. The final products were obtained by
drying the precipitates under vacuum for 24 h.
Fabrication of Core-Cross-Linked Prodrug NPs with Control-
lable Drug Content. A typical procedure of the RAFT dispersion
polymerization for fabrication of the prodrug NPs is as follows: PEG-
b-P(MEO2MA-co-CPTM) (145 mg, 0.01 mmol), AIBN (0.328 mg, 2
× 10−3 mmol), BzMA (70 mg, 0.40 mmol), CBMA (29 mg, 0.10
mmol), and ethanol/water (2.44 g, the mass ratio of 7/3) were added
into a 5 mL glass tube with a magnetic bar. The tube was sealed under
vacuum after three freeze−evacuate−thaw cycles. After the polymer-
ization was conducted at 70 °C for 24 h, the reaction was quenched by
rapidly cooling to room temperature. For the RAFT dispersion
polymerizations using other three macro-CTAs, the same procedures,
the same total solids content (10%), and the same molar ratio of
AIBN/PEG-b-P(MEO2MA-co-CPTM) (1/5) were used.
Characterization. The 1H NMR spectra were measured on a
Bruker DMX300 spectrometer (300 MHz) using CDCl3 as the solvent
and tetra-methylsilane as the internal reference. Molecular weight
(Mn) and Mw/Mn were measured using a Waters 150C gel permeation
chromatography (GPC), equipped with RI 2414 detector (set as 30
°C) and two Ultrastyragel columns in series, monodispersed
polystyrene standards were used in calibration, and tetrahydrofuran
(THF) was used as eluent at a flow rate of 1.0 mL/min. Transmission
electron microscope (TEM) characterizations were conducted on a
Hitachi H-800 electron microscope at an accelerating voltage of 100
kV. Dynamic light scattering characterizations were performed on a
Laser Light Scattering (LLS) spectrometer, Zetasizer Nano ZS90
(Malven Instruments Ltd., Malvern, U.K.), equipped with a He−Ne
Laser (4.0 mW, 633 nm) at 25 °C and a fixed angle of 90°. UV−vis
spectra were obtained by a TU-1901 UV−vis spectrophotometer. The
fluorescence spectra were acquired on a Shimadzu RF-5301PC
luminescence spectrometer, and the slit widths were both set at 10
nm for excitation and for emission. Confocal laser scanning
microscopy (CLSM) observations were conducted using a Leica
TCS SP5 microscope.
RESULTS AND DISCUSSION
■
Preparation and Characterization of Prodrug Diblock
Copolymer PEG-b-P(MEO2MA-co-CPTM). To avoid de-
struction of the dithiobenzoate during the reaction between
PEG113-NH2 and 4-cyano-4-((phenylcarbono-thioyl)thio) pen-
tanoic acid (CPDB), a feasible and highly efficient method is
the reaction of succinimide activated ester with amino group of
PEG113-NH2 at mild condition.42 Thus, the first step is the
synthesis of the succinimide-modified cyanopentanoate dithio-
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benzoate (SCPDB) by the esterification reaction of N-
1
hydroxysuccinimide and CPDB. The H NMR spectrum of
the SCPDB is shown in Figure S1. The aromatic proton signals
appear at δ = 7.3−8.0 ppm (signals d−f). Signals b (δ = 2.5−
2.8 ppm, 3.0 ppm) and c (δ = 1.9 ppm) are, respectively,
attributed to the methylene protons of pentanoic acid and
methyl protons adjacent to the nitrile group. The signal at δ 2.9
ppm (a) is assigned to the methylene protons of succinimide,
and quantification ratios of the signals’ integral values are in
accordance with the structures of SCPDB. All these data
support successful preparation of the SCPDB. Then the
PEG113-CPDB macro-RAFT agent was prepared by the
reaction of SCPDB with the methoxypolyethylene glycol
amine (PEG113-NH2), which has been proved to be a superior
method for synthesis of the PEG113-CPDB owing to high extent
of end-group modification.42,43 Figure 1 shows the H NMR
1
spectrum of the resultant PEG113-CPDB. The proton signals at
δ = 7.3−8.0 ppm (f−h), 2.3−2.7 ppm (d), and 1.9 ppm (e) are,
respectively, attributed to the aromatic protons, two methylene
protons, and methyl protons of the CPDB unit; the ethylene
and methyl proton signals of the PEG113 appear at δ = 3.67 (b)
and 3.4 ppm (a), respectively. The signal at δ = 6.5 ppm (c) is
ascribed to the amido proton, so, the amidation reaction
between the N-hydroxysuccinimide-activated ester and the
amino group of PEG113-NH2 is successful. Based on integral
values of the aromatic proton signals (f−h) and the PEG
proton signal (b), an average degree of the dithiobenzoate
functionalization is 96%, and the highly capping efficiency is
consistent with the previous reports.42,43
1
Figure 2. H NMR spectra of prodrug diblock copolymers, PEG-b-
P(MEO2MA-co-CPTM) prepared from the copolymerizations with
various feed molar ratios of (A) CPTM/MEO2MA = 1/2, (B) CPTM/
MEO2MA = 1/4, (C) CPTM/MEO2MA = 1/8.
Camptothecin (CPT), which is isolated from Chinese
Camptotheca acuminata (happy tree), was selected as the
model drug due to its significant anticancer activity. For
preparation of the reductive-sensitive prodrug through
copolymerization, the first step is synthesis of the CPT-
contained monomer with disulfide (CPTM), which was
synthesized according to the procedure described in the
literature.44 All proton signals of the CPTM are marked in
CPT contents in the PEG-b-P(MEO2MA-co-CPTM) increase
from 13.4 to 22.7%, and then to 36.3% (Table 1). GPC traces
of the PEG-b-P(MEO2MA-co-CPTM)s shown in Figure 3
reveal that the three prodrug macro-CTAs with different drug
contents have similar molecular weights. For the sake of brevity,
PEG-b-P(MEO2MA-co-CPTM)s synthesized with the feed
molar ratios of CPTM/MEO2MA = 1/8, 1/4, and 1/2 are
simply denoted as macro-CTA1, macro-CTA2, and macro-
CTA3, respectively.
1
the H NMR spectrum of Figure S2, which supports that
synthesis of the CPTM is successful.
Fabrication of Prodrug NPs with Controllable Drug
Content and Superior Structural Stability. The core-cross-
linked prodrug NPs were prepared by RAFT dispersion
polymerization of CBMA and BzMA in a mixture of ethanol
and water. The first step is synthesis of cross-linker CBMA,
which was synthesized according to our previous reported
The prodrug diblock copolymer, PEG-b-P(MEO2MA-co-
CPTM) was synthesized by copolymerization of CPTM and
MEO2MA using PEG113-CPDB as macro-RAFT agent, as
shown in Scheme S1. The drug content in the diblock
copolymers can be modulated by the feed molar ratio of
CPTM/MEO2MA. Herein, the feed molar ratios of CPTM/
MEO2MA = 1/2, 1/4 and 1/8 with a constant total molar
content were used to prepare the prodrug diblock copolymers.
method,45 and its structure was characterized by H NMR.
1
1
Successful synthesis of CBMA is supported by the H NMR
spectrum (Figure S3): vinyl proton signals appear at δ = 5.45
and 5.72 ppm (b); ethylene proton signals next to imide
nitrogen and adjacent to sulfur, respectively, appear at 3.65 (d)
and 2.92 (e) ppm; methyl proton signal appears at 1.95 ppm
(a). Then all three macro-CTAs (macro-CTA1, macro-CTA2,
and macro-CTA3) were used to fabricate prodrug NPs via one-
pot RAFT dispersion polymerization of BzMA/CBMA with a
feed molar ratio of 8/2 (Scheme 1). A mixture of ethanol/water
with a mass ratio of 7/3 was used as the reaction media. Both of
the monomers (BzMA and CBMA) and the macro-CTA (PEG-
b-P(MEO2MA-co-CPTM)) are well soluble in the reaction
media, but the in situ formed P(BzMA-co-CBMA) by
polymerization has poor solubility in the mixture. So the
PISA occurs in this system. Because the drug was conjugated on
the macro-CTA, it is envisioned that drug loading was achieved
during the NPs formation. When the RAFT dispersion
polymerizations were, respectively, conducted using macro-
1
Figure 2 shows the H NMR spectra of the resultant PEG-b-
P(MEO2MA-co-CPTM)s. The proton signal at δ = 4.1 ppm
(n), which is assigned to the ester methylene of MEO2MA,
increases clearly with the feed molar ratio of CPTM/MEO2MA
decreasing from 1/2 to 1/4 and to 1/8. The actual molar ratio
of CPTM/MEO2MA in the PEG-b-P(MEO2MA-co-CPTM)
were calculated according to the 1H NMR spectra by
comparing the integral values of the ester proton signal (l) of
the CPTM units to that of the ester proton signal (n) of the
MEO2MA units, and the results are listed in Table 1. It can be
seen that the actual molar ratios of CPTM/MEO2MA in the
resultant PEG-b-P(MEO2MA-co-CPTM) are approximately
consistent with the feed molar ratio, so, the drug content in
the prodrug can be controlled by the feed molar ratio of
CPTM/MEO2MA. When the feed molar ratios of CPTM/
MEO2MA increase from 1/8 to 1/4 and to 1/2, the resultant
D
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Table 1. Summary of the Feed Molar Ratio of CPTM/MEO2MA for Preparation of Prodrug Macro-CTAs, the Actual Molar
Ratio of CPTM/MEO2MA, the CPT Content in the Resultant Macro-CTAs, the CPT Content in the Resultant Prodrug NPs,
and the Diameter/PDI of the Resultant Prodrug NPs
b
feed molar ratio of
CPTM/MEO2MA
molar ratio of CPTM/MEO2MA in
the macro-CTAs
CPT content in the prodrug
macro-CTAs/%
CPT content in the
prodrug NPs/%
diameter/nm
(PDI)
a
sample
prodrug
NPs-1
1/8
1/4
1/2
0/1
1/9.02
1/4.65
1/2.32
0
13.4
22.7
36.3
0
4.2
7.4
12.8
0
36.3 (0.119)
37.2 (0.111)
36.8 (0.095)
36.6 (0.087)
prodrug
NPs-2
prodrug
NPs-3
NPs-4
a
The prodrug NPs-1, -2, and -3 were, respectively, fabricated using macro-CTA1, macro-CTA2, and macro-CTA3. NPs-4 are drug-free spherical
b
micelles that were fabricated using PEG-b-PMEO2MA (macro-CTA4). The intensity-average diameter and polydispersity of the NPs determined by
DLS.
shown in Figure S5. The morphology of prodrug NPs in THF
still remained very well (Figure S5), which indicates superior
structural stability of the prodrug NPs due to the core-cross-
linking during the PISA. DLS diameters of the prodrug NPs in
THF are nearly twice as big as them in water (Figure S6),
which indicates that the core of prodrug NPs are swollen
instead of dissolving in THF due to the core-cross-linking
structure. All the above results verify that the prodrug NPs with
robust structure are efficiently fabricated via PISA strategy and
the drug contents in the resultant prodrug NPs can be
accurately modulated via adjusting the feed molar ratio of
MEO2MA/CPTM during preparation of the prodrug macro-
CTA (PEG-b-(PMEO2MA-co-CPTM)).
Figure 3. GPC traces of the PEG-CPDB (black line) and the PEG-b-
P(MEO2MA-co-CPTM) fabricated with various feed molar ratios of
CPTM/MEO2MA: 1/8 (blue line), 1/4 (green line), and 1/2 (red
line).
Redox-Regulated Drug Release. The proposed release
mechanism of parent drug CPT from the NPs upon reductive
milieu is shown in Scheme 2.44 In vitro drug release
experiments for the obtained prodrug NPs with different drug
contents were performed at various concentrations of the GSH
(0.01, 5, and 10 mM). High performance liquid chromatog-
raphy (HPLC) was used to characterize the released drug and
the pure CPT. Figure S7 shows that the pure CPT and the
released drug from NPs-3 have the same retention time, which
illustrates that the released CPT has the same structure of its
parent CPT. The cumulative CPT release was evaluated using
the fluorescence spectrometer. In the presence of 0.01 mM of
GSH, less than 5% cumulative CPT was released upon
incubation for 48 h, as shown in Figures 5 and S8, which
reveals a minimal premature drug release during blood
circulation. After incubation for 48 h in the presence of 5 or
10 mM GSH, higher cumulative CPT release was achieved:
exceeding 35% CPT release for 5 mM GSH and 42% for 10
mM GSH (Figures 5 and S8), which is reasonable because the
cleavage rate of the disulfide bond increases with concentration
CTA1, macro-CTA2, and macro-CTA3, almost complete
monomer conversions (>99%) were achieved within 24 h of
polymerization, as judged by 1H NMR spectra (the vinyl
proton signals of the monomers are almost invisible, the data
are not shown), and NPs with the similar diameter were
obtained as characterized by DLS and TEM (Table 1 and
Figure 4). Due to the superior hydrophilic PEG blocks on the
Figure 4. TEM images of the prodrug NPs fabricated by RAFT
dispersion polymerizations using different PEG-b-(PMEO2MA-co-
CPTM)s as the macro-CTA: (A) macro-CTA1; (B) macro-CTA2;
(C) macro-CTA3.
shell, these nanoparticles also dispersed well in water. TEM
images in Figure S4 show that shape and dispersity of the
nanoparticles are not affected by their transfer from ethanol/
water (7/3) to pure water. The parent CPT content in the
resultant prodrug NPs are modulated by using different macro-
CTAs, which can be calculated to be 4.2, 7.4, and 12.8% when
the macro-CTA1, macro-CTA2, and macro-CTA3 are used,
respectively. TEM images of the prodrug NPs in tetrahydrofur-
an (THF, which is a good solvent for all of the blocks) are
Figure 5. In vitro CPT release profiles of the prodrug NP-3 at various
concentrations of the GSH.
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Figure 6. In vitro cytotoxicity of the NPs with different concentrations of the NPs: (A) the drug-free NPs (fabricated using macro-CTA4), (B) the
NPs fabricated using prodrug macro-CTAs (black line: macro-CTA1, red line: macro-CTA2, green line: macro-CTA3).
increase of the GSH. All three CPT release profiles in Figures 5
and S8 display two stages of the CPT release: faster release rate
within an initial 5−7 h and then the release rate decreasing,
such as, for the drug release of prodrug NPs-3 in the presence
of 10 mM GSH, the release rates before and after 7 h are 36.7
and 7.5 μg/h, respectively. Thus, upon cell internalization, the
drug release can be efficiently enhanced subjecting to high
concentration of GSH. As previous reports,19,40 high
concentration of GSH also induced cleavage of the reductive-
sensitive cross-linker and then dissociation of the NPs.
However, due to the hydrophobic cores of the NPs herein,
the GSH is difficult to diffuse into the cores of the NPs in
aqueous solution leading to very slow degradation of the core
cross-linker. When the degradation of NPs-3 was conducted in
a mixed solvent of THF/water (9/1, v/v, a little of water is used
to dissolve the GSH), which is a good solvent for both of the
blocks in the NPs. The degradation of NPs was observed, and
the diameter of the NPs-3 reduced from 67.1 to 4.3 nm
(detected by LLS) after stirring for 24 h in the presence of 10
mM GSH. This is because the GSH is easy to diffuse into the
swollen core of NPs resulted in fast dissociation of the NPs. It
is reasonable to speculate that, in the high dilution environ-
ment, such as in body fluids, the GSH can also diffuse into the
core of NPs and induced the degradation of them.
to evaluate cytotoxicity of the NPs directly after the
polymerization without any further purification procedures.
Figure 6A shows that the NPs fabricated using macro-CTA4 are
almost noncytotoxic up to a concentration of 1000 μg/mL,
which is in accordance with the well-recognized biocompati-
bility of PEG.5,6 Meanwhile, the above results indicate that the
ethanol used in the PISA has negligible cytotoxicity due to the
very low content after high dilution (Note: the solid content of
the obtained NPs is 100 mg/mL, and the highest NP
concentration for cytotoxicity evaluation is 1 mg/mL, so, at
least 100 times dilution of the obtained NP dispersions by the
PBS buffer is necessary for the cytotoxicity evaluation). The
prodrug NPs, which were directly obtained from the RAFT
dispersion polymerization using the macro-CTA1, the macro-
CTA2, and the macro-CTA3 without any further purification
procedure, were added to HeLa cells, and the results are shown
in Figure 6B. Dramatically decreased cell viability was observed
as the concentration increasing of all the three prodrug NPs
(Figure 6B). Because of the three prodrug NPs having similar
diameter (Table 1 and Figure 4), influence of the NPs size on
their anticancer activities can be ignored. The IC50s (inhibitory
concentration for 50% cell viability) of the three prodrug NPs
are 145 μg/mL for prodrug NPs-1 (equiv 6.09 μg/mL of CPT),
85 μg/mL for prodrug NPs-2 (equiv 6.29 μg/mL of CPT), and
50 μg/mL for prodrug NPs-3 (equiv 6.40 μg/mL of CPT),
respectively; thus, the cytotoxicity of three prodrug NPs has the
following order: prodrug NPs-1 (CPT content: 4.2%) <
prodrug NPs-2 (7.4%) < prodrug NPs-3 (12.8%), which is the
same order with increase of the CPT contents. However, the
CPT doses at the IC50 are very close to each other (6.09−6.40
μg/mL) for the three prodrug NPs, which further indicate that
the cytotoxicity are majorly induced by CPT, and the NPs with
high CPT content release more CPT molecules in the same
duration resulting in high cytotoxicity, that is, they exhibit more
excellent anticancer efficiency. So, the less prodrug NPs are
needed to kill the HeLa cells, and less polymeric matrix are
remained after drug delivery, which is more appreciate for
cancer chemotherapy concerning the long-term toxicity of
polymeric nanocarriers in the organism. The above results
reveal that all the three prodrug NPs exhibit excellent
performance of intracellular drug release.
Cytotoxicity Evaluation. In vitro cytotoxicity was
evaluated against HeLa cells using MTT assay. Before studying
cytotoxicity of the prodrug NPs, it is necessary to evaluate the
cytotoxicity of the nanocarriers without drug conjugation
because well-biocompatible nanocarriers are highly desired for
reducing the side effect in drug delivery. The macro-CTA4
without CPT-conjugation was synthesized by RAFT polymer-
ization of MEO2MA using PEG-CPDB as the macro RAFT
1
agent. H NMR spectrum of the resultant polymer shown in
Figure S9 demonstrates that successful chain extension of the
PEG-CPDB was realized to get diblock copolymer PEG-b-
PMEO2MA (macro-CTA4). As previously reported,28 the
molecular weight of macro-CTA has significant influence on
the size of the NPs fabricated through PISA strategy. For
fabricating nanocarriers (without drug conjugation) with similar
size to the above-mentioned prodrug NPs, it is expected to get
macro-CTA4 with the molecular weight similar to the other
three macro-CTAs. For this purpose, the same polymerization
conditions were conducted. Macro-CTA4 with Mn = 15600 was
obtained (Figure S10), which is approximately equal to the
other three macro-CTAs (Figure 3). As expected, the NPs
fabricated using macro-CTA4 have similar size to the other
three prodrug NPs (Table 1, Figures 4 and S11), which is
reasonable owing to the similar molecular weights of the macro-
CTAs and the core-forming block. Then MTT assay was used
To further investigate the intracellular drug release behaviors
of the prodrug NPs, confocal laser scanning microscopy
(CLSM) was used to study the internalization of the prodrug
NPs by HeLa cells. The CPT emission was used directly to
visualize cellular uptake of the NPs without additional
fluorescence labeling. So the amount of CPT-conjugated NPs
internalized by HeLa cells should in principle be proportional
to the fluorescence intensity. The three prodrug NPs with
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equivalent CPT content (10 μg/mL) were used to incubate
HeLa cells. As expected, no obvious difference on cell
internalization of the three prodrug NPs at all the incubation
time (4, 8, and 16 h) were observed due to similar size of the
NPs (Figure 7). After incubation with the prodrug NPs for 4 h,
efficient approach for fabrication of the polymeric drug delivery
systems in cancer chemotherapy.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.6b00819.
■
S
1H NMR of the succinimide-modified cyanopentanoate
dithiobenzoate (SCPDB), reduction-cleavable campto-
thecin (CPT) prodrug monomer (CPTM), cross-linker
(CBMA), and the drug-free macro-CTA PEG-b-
PMEO2MA, DLS of the prodrug NPs, a part of TEM
images, GPC trace, drug release profiles (Figures S1−
S11), and the scheme of synthesize prodrug macro-CTA
PEG-b-P(MEO2MA-co-CPTM) (Scheme S1; PDF).
AUTHOR INFORMATION
■
Figure 7. CLSM images recorded for HeLa cells after incubation for 4,
8, and 16 h with the (A) prodrug NPs-1, (B) prodrug NPs-2, and (C)
prodrug NPs-3.
Corresponding Authors
*E-mail: hongcy@ustc.edu.cn.
*E-mail: pcy@ustc.edu.cn.
Notes
The authors declare no competing financial interest.
only weak blue fluorescence was observed, but the fluorescence
intensity remarkably increased after 8 h of incubation, which
reveals excellent cell internalization of the prodrug NPs. It is
crucial for parent CPT to be released from the prodrug NPs
and then the released CPT molecules are accumulated in the
cell nucleus for anticancer activity via interaction with genomic
DNA to induce pharmacological responses. Significant blue
fluorescence is observed within the nucleus of HeLa cells after
incubation of 16 h with all the three prodrug NPs, which
indicates that the highly reductive intracellular microenviron-
ment should be responsible for the efficient CPT cleavage from
the prodrug NPs and nuclear accumulation of the drugs (Figure
7). Thus, we report a superior strategy of fabricating the
prodrug NPs for effectively anticancer chemotherapy.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support from the
National Natural Science Foundation of China under Contract
Nos. 21525420 and 21374107, the Fundamental Research
Funds for the Central Universities (WK 2060200012 and WK
2060200015), and China Postdoctoral Science Foundation
(BH2060000011).
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