Glutathione and endosomal pH-responsive hybrid vesicles fabricated by zwitterionic polymer block poly(L-aspartic acid) as a smart anticancer delivery platform

Article abstract of DOI:10.1016/j.reactfunctpolym.2017.07.010

Zwitterionic hybrid block copolymer based nanocarriers are ideal candidates for drug delivery applications due the higher resistance to nonspecific protein adsorption in complex media compared to nonionic polymers. Especially, zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) p(MPC) based nanocarriers can maintain its stability during circulation in complex media, such as serum. Thus, a series of bioreducible and pH-responsive zwitterionic/amphiphilic block copolymers, poly(2-methacryloyloxyethyl phosphorylcholine)₅₀-block-poly(L-aspartic acid)_n (p(MPC)₅₀–b–p(AA)_n) (n = 10, 25, 50, 75), bearing a degradable disulfide linker have been synthesized and exploited as dual-stimuli-responsive drug delivery vehicle of the chemotherapeutic drug, doxorubicin (Dox). Dox was successfully loaded into uniform vesicles (～ 100 nm) fabricated from p(MPC)₅₀–b–p(AA)_n and the release performance was investigated under different pH conditions and with a range of concentrations of the reducing agent, 1,4-dithiothreitol (DTT). At physiological conditions, increasing concentrations of DTT resulted in faster Dox release from vesicles. Dox release at elevated DTT concentrations was more effective at pH 5.5 than at pH 7.5. Blank vesicles were non-toxic over a wide concentration range when tested in normal cell lines (0.01–100 μg/mL). Vesicles efficiently encapsulated Dox and the dual stimuli-responsive disassembly results demonstrated controlled and sustained release of Dox tin 4T1 cancer cells to confer dose-dependent cytotoxicity. Thus, the bioreducible and pH sensitive vesicles appear to be a promising theranostic platform for drug delivery applications.

Full text of DOI:10.1016/j.reactfunctpolym.2017.07.010

Accepted Manuscript
Glutathione and endosomal pH-responsive hybrid vesicles
fabricated by zwitterionic polymer block poly(l-aspartic acid) as a
smart anticancer delivery platform
Renjith P. Johnson, Saji Uthaman, Rimesh Augustine, Yu Zhang,
Hwa Jin, Chang In Choi, In-Kyu Park, Il Kim
PII:
S1381-5148(17)30138-4
DOI:
Reference:
doi: 10.1016/j.reactfunctpolym.2017.07.010
REACT 3884
To appear in:
Reactive and Functional Polymers
Received date:
Revised date:
Accepted date:
22 May 2017
10 July 2017
29 July 2017
Please cite this article as: Renjith P. Johnson, Saji Uthaman, Rimesh Augustine, Yu Zhang,
Hwa Jin, Chang In Choi, In-Kyu Park, Il Kim , Glutathione and endosomal pH-responsive
hybrid vesicles fabricated by zwitterionic polymer block poly(l-aspartic acid) as a smart
anticancer delivery platform, Reactive and Functional Polymers (2017), doi: 10.1016/
j.reactfunctpolym.2017.07.010
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ACCEPTED MANUSCRIPT
Glutathione and endosomal pH-responsive hybrid vesicles fabricated by
zwitterionic polymer block poly(L-aspartic acid) as a smart anticancer
delivery platform
Renjith P. Johnsona,1, Saji Uthamanb, Rimesh Augustinea, Yu Zhanga, Hwa Jina, Chang In Choic,
In-Kyu Parkb, Il Kima,*
a BK21 PLUS Centre for Advanced Chemical Technology, Department of Polymer Science and
Engineering, Pusan National University, Busandaehak-ro 63, Geumjeong-gu, Busan, 46241,
Republic of Korea
b Department of Biomedical Science and BK 21 PLUS Center for Creative Biomedical Scientists,
Chonnam National University Medical School, 160 Baekseo-ro, Gwanju, 501-746, Republic of
Korea.
c Department of Surgery, Medical Research Institute, Pusan National University Hospital, Busan
602-739, South Korea
_______________________
1 Present address: Yenepoya Research Centre, Yenepoya University, Mangalore, India
* Corresponding author. Tel: +82-51-510-2466. E-mail: ilkim@pusan.ac.kr
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Abstract
Zwitterionic hybrid block copolymer based nanocarriers are ideal candidates for drug delivery
applications due the higher resistance to nonspecific protein adsorption in complex media
compared to nonionic polymers. Especially, zwitterionic poly(2-methacryloyloxyethyl
phosphorylcholine) p(MPC) based nanocarriers can maintain its stability during circulation in
complex media, such as serum. Thus, a series of bioreducible and pH-responsive
zwitterionic/amphiphilic block copolymers, poly(2-methacryloyloxyethyl phosphorylcholine)50-
block-poly(L-aspartic acid)n (p(MPC)50–b–p(AA)n) (n = 10, 25, 50, 75), bearing a degradable
disulfide linker have been synthesized and exploited as dual-stimuli-responsive drug delivery
vehicle of the chemotherapeutic drug, doxorubicin (Dox). Dox was successfully loaded into
uniform vesicles (∼100 nm) fabricated from p(MPC)50–b–p(AA)n and the release performance
was investigated under different pH conditions and with a range of concentrations of the
reducing agent, 1,4-dithiothreitol (DTT). At physiological conditions, increasing concentrations
of DTT resulted in faster Dox release from vesicles. Dox release at elevated DTT concentrations
was more effective at pH 5.5 than at pH 7.5. Blank vesicles were non-toxic over a wide
concentration range when tested in normal cell lines (0.01–100 µg/mL). Vesicles efficiently
encapsulated Dox and the dual stimuli-responsive disassembly results demonstrated controlled
and sustained release of Dox tin 4T1 cancer cells to confer dose-dependent cytotoxicity. Thus,
the bioreducible and pH sensitive vesicles appear to be a promising theranostic platform for drug
delivery applications.
Keywords: Biodegradation, Block copolymer, Drug delivery, Polypeptide, Responsive polymer
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1. Introduction
Chemotherapeutic drug delivery vehicles such as polymer drug conjugates [1], liposomes [2],
polymeric micelles [3–5], and polymersomes [6, 7] (vesicles of synthetic amphiphilic block
copolymers) have been widely investigated in recent decades for tumor-targeted treatments.
Generally, these nano-vehicles enhance the solubility and bioavailability of drugs, allowing the
drug to act directly at the target site of interest. By avoiding rapid clearance by the
reticuloendothelial system (RES), these carriers can have a long circulation time. This
contributes to accumulation at the tumor site through the enhanced permeability and retention
(EPR) effect that occurs in response to abnormalities of tumor blood and the lymphatic
vasculature [8, 9]. However, several drawbacks to applications of these carriers still persist,
including systemic toxicity, reduced intracellular uptake, and poor endosomal escape after the
cellular entry [10–12].
"Smart" polymeric nano-vehicles can respond to physiological conditions in vivo, such as pH
[13, 14], temperature [15], certain enzymes [16], redox substances [17], and even multiple stimuli.
This attracted great attention in the drug delivery domain. Undoubtedly, pH is one of the most
frequently exploited physiological stimuli owing to the significant pH difference between blood
and extracellular matrices and that of intracellular compartments [18]. To date, several pH-
responsive delivery vehicles, including micelles or vesicles, have been developed to target tumor
tissue or cellular compartments [3, 6]. "Titratable" groups such as imidazoles, tertiary amines,
and carboxylic acid-containing amphiphilic block copolymer assemblies can exhibit a "proton-
sponge" effect in acidic environments and these have been designed for intracellular-targeted
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drug release [19–21]. Recently, we developed poly(L-histidine)-based hybrid materials for pH or
pH/temperature dual responsive nano-vehicles for cancer therapy [4, 5, 22, 23].
In recent decades, parallel progress has been made by several research groups in the design
of bioreducible nano-carriers for triggered intracellular release of anticancer drugs [6, 24]. The
basic principle behind the use of bioreducible carriers is the cleavage of the disulfide (SS) bond
of the molecular structures, either when located in the main chain, at the side chain, or as a cross-
linker, upon exposure to reductive intracellular environments. The abundance of the SS-reducing
biological thiol, glutathione tripeptide (L-γ-glutamyl-L-cysteinyl-glycine; GSH) is much higher
in the cytosol than in the blood and other extracellular fluids (2−10 mM in cytosol vs. 2−20 μM
in extracellular fluids) [25]. Thus, the SS bonds are sufficiently stable in the circulating blood
and in the extracellular environment, but can undergo rapid cleavage through the thiol–disulfide
exchange reaction, at a time scale of minutes to hours, under more reductive intracellular
environment [26]. Additionally, tumor tissues are highly reducing and hypoxic when compared
with normal tissues [27] and contain at least 4-fold higher concentrations of GSH.
The characteristic acidic and reductive tumor environment demands the design and synthesis
of multi-stimuli responsive vehicles for attaining an improved therapeutic effect. Even though
liposomes and micelles have been investigated as major platforms for the delivery of anticancer
drugs and imaging agents, it is recognized that pure liposomes suffer from very rapid blood
clearance in vivo by the RES, unless a surface modification approach is applied. Recently,
polymer vesicles have attracted increasing interest owing to their cell-and virus-mimicking
dimensions, function, and their ability to overcome the chemical and physical limits of liposomes
and micelles [28, 29]. To date, the syntheses of several amphiphilic block copolymers and the
fabrication of their vesicles have been reported. The first-generation polymer vesicles [30–32]
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were derived from block copolymers that were not completely biodegradable, which was a major
drawback limiting their biomedical utility. Over the last decade, pure polypeptidic [33] and
polypeptide-hybrid vesicles or hydrogels have emerged, and these have been investigated for
delivery of the anticancer drug doxorubicin hydrochloride (Dox) [34–39].
In the past decades, polyethylene glycol (PEG) was the most common choice as a
hydrophilic biocompatible segment in designing various polymeric drug carriers. However, PEG
with lower molecular weight (i.e., less than 3000) was cytotoxic in the body [40]. Recently,
poly(2-hydroxyethyl methacrylate) [41] and poly[2-(methacryloyloxy)ethyl phosphorylcholine]
[p(MPC)] were emerged as alternatives for PEG. Interestingly, MPC is a zwitterionic,
hemocompatible and highly hydrophilic monomer, which mimicks the phospholipid headgroups
located on the surface of cell membranes, remarkably resistant to protein adsorption as well as
bacterial/cellular adhesion [42]. Additionally, p(MPC) has been widely implemented in surface
modification as biomaterials that exhibit exceptional hydrophilicity, biocompatibility, and
antifouling properties [43]. Thus, in order to overcome the limitation of liposomes and micelles
by fabricating pH/reduction dual-responsive vesicles from block copolymer composed of
p(MPC) and relatively hydrophobic and pH-responsive poly(L-aspartic acid) [p(AA)] is designed.
Herein, we have synthesized a block copolymer poly(2-(methacryloyloxy)ethyl
phosphorylcholine)50–b–poly(L-aspartic acid)n (p(MPC)50-b-p(AA)n) (n= 10, 25, 50, 75) bearing
a redox responsive disulfide (–SS–) linker for the triggered delivery of Dox. The synthesis
employed reversible addition fragmentation chain transfer (RAFT) polymerization, ring-opening
polymerization (ROP) of β-benzyl-L-aspartate N-carboxyanhydride (BLA–NCA) and click-
cycloaddition reactions. pH and reduction dual-responsive vesicles for encapsulation of Dox
were fabricated. The successful redox and pH-responsive in vitro release of Dox was
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investigated and the cytotoxicity of the vesicles assessed in mouse embryonic fibroblast (NIH
3T3) and mouse mammary tumor (4T1) cell lines; the anticancer effect of the released Dox was
confirmed in 4T1 cell lines.
2. Experimental
2.1. Materials
4-(Dimethylamino)pyridine (DMAP, 99%), N,N ′ -dicyclohexylcarbodiimide (DCC,
99.0%), 2,2 ′ -azobis(2-methylpropionitrile (AIBN 98%), 3-bromo-1-propanol, 2-bromo-2-
phenylacetic acid, sodium thiomethoxide (NaSCH3-21% in water), carbon disulfide CS2, 99.9%),
2-methacryloyloxyethyl phosphorylcholine (MPC, 97%), 1,1′-carbonyldiimidazole, propargyl
alcohol (99%), cystamine dihydrochloride (96%), β-benzyl-L-aspartate (BLA) and anhydrous
N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich. Dox was purchased from
Lancrix Chemicals (Shanghai, China). Hexylamine (99.9%), trifluoroacetic acid (99%), HBr (33
wt% in acetic acid), sodium azide, and triphosgene (>98%) were purchased from TCI (Tokyo,
Japan). Tetrahydrofuran (THF), methanol, chloroform, and dichloromethane (DCM) were dried
over calcium hydride and distilled under reduced pressure. All other reagent-grade chemicals
were purchased from Daejung Chemicals (Siheung, Korea) and used without further purification.
Deionized (DI) water (purified to a resistance of 18 MΩ (Milli−Q Reagent Water System,
Millipore Corp., Billerica, MA, USA) was used in all solution preparations, polymer isolations,
and necessary reactions.
2.2. General methods
All column chromatography purifications were performed using a Combi-Flash companion
1
purification system (Teledyne ISCO) with silica gel of 300-400 mesh. H-NMR (400 MHz) and
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13C-NMR (100 MHz) spectra were recorded on a Varian INOVA 400 NMR spectrometer.
Chemical shifts were reported in parts per million (ppm) relative to the residual solvent peaks as
1
internal standard. Peak multiplicities in H-NMR spectra are abbreviated as s (singlet), d
(doublet), t (triplet), m (multiplet), and br (broad). Fourier-transform infrared (FT–IR) spectra
were recorded using a Shimadzu IR prestige 21 spectrometer on KBr discs over the range of
3500–500 cm−1 at room temperature. UV−vis experiments conducted on a shimadzu UV-1650
PC. The molecular weight (MW) and polydispersity index (Đ) of the polymers were measured
using a Waters GPC system, which was equipped with a Waters 1515 HPLC solvent pump, a
Waters 2414 refractive index detector, and three Waters Styragel High-Resolution columns (HR4,
HR2, HR1, effective molecular weight range 5000–500,000, 500–20,000, and 100–5000 g mol−1,
respectively) at 40 °C with HPLC-grade DMF containing 0.1 N LiBr as eluent at a flow rate of
1.0 mL/min. Monodispersed polystyrenes were used to generate the calibration curve. DLS
measurements were performed with a high-performance Zetasizer Nano ZS90 (Malvern
Instruments, Ltd., U.K.). Block copolymer solutions (2 mg·mL−1) were filtered through a 0.5 µM
filter prior to use. Transmission electron microscopy (TEM) was performed using a JEOL–
1299EX TEM at an accelerating voltage of 80 keV.
2.3. Synthesis and polymerization. The general synthetic and polymerization procedures are
presented in Scheme 1.
2.3.1. Synthesis of S-azidopropanoxycarbonylphenylmethyl trithiocarbonate (CTA–N3)
For the RAFT polymerization of MPC, an azide-terminated chain transfer agent was
synthesized by following the report of Ladmiral and coworkers [42,44]. The spectroscopic
characterizations of the CTA–N3 are provided in Fig. S1 of the Supplementary material (SM).
2.3.2. Synthesis of propargyl ester of carbonyl imidazole (PPA–CIM).
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PPA–CIM was synthesized as described previously [45]. 1,1’-Carbonyldiimidazole (7.3 g,
45 mmol) and 50 mL DCM were added into a dry round-bottom flask. Then, propargyl alcohol
(1.45 mL, 25 mmol) was added dropwise. The obtained clear solution was stirred at room
temperature for 1 h, and washed with water (3×70 mL). The product was dried over magnesium
1
sulfate, and evaporated to obtain PPA-CIM as an off-white solid. Yield: 80%. H-NMR (400
MHz, CDCl3, 298 K): δ (ppm) 8.19 (s, 1H, NCHN), 7.45 (s, 1H, NCHCHN), 7.10 (s, 1H,
NCHCHN), 5.0 (s, 2H, OCH2C), and 2.63 (s, 1H, CCH).
2.3.3. Synthesis of ((Propargyl carbamate)ethyl) Dithioethylamine (PPA–CAM).
Cystamine dihydrochloride was neutralized by 4 M NaOH and extracted with DCM to yield
cystamine. PPA–CIM (1.44 g, 9.58 mmol) and cystamine (2.28 g, 15 mmol) were dissolved in
30 mL DCM and subsequently stirred at room temperature for 24 h. After evaporation of DCM,
25 mL of 1.0 M NaH2PO4 (pH 4.0) was added. This aqueous solution was extracted with diethyl
ether (20 mL, 5 times) to remove the di-alkyne compound bis[(propargyl carbamate)ethyl]
disulfide. The aqueous phase was basified with 1.0 M NaOH to pH 9.0, extracted with ethyl
acetate (15 mL, 5 times), and dried over anhydrous magnesium sulfate. Rotary evaporation
resulted in PPA–CAM as a pale yellow viscous liquid [43,45]. Yield: 42%. 1H-NMR (400 MHz,
CDCl3, 298 K): δ (ppm) 2.45 (s, 1H, CH≡C), 2.75 (m, 4H, S–CH2–C), 2.98 (m, 2H, NH2–CH2–
C), 3.50 (m, 2H, NH–CH2–C), 4.37 (b, 2H, NH2–C), 4.64 (s, 2H, O-CH2-C≡C), 5.58 (s, 1H,
NH–C=O). The spectroscopic characterizations of the PPA–CAM are provided in Fig. S2 of SM.
2.3.4. RAFT Polymerization of MPC from CTA–N3.
To a 10-mL Schlenk tube equipped with a magnetic stir bar, the following were added; MPC
(1.48 g, 5 mmol), CTA–N3 (34 mg, 0.1 mmol), AIBN (8 mg, 0.05 mmol), and methanol (5 mL).
The [monomer]:[CTA]:[AIBN] ratio was 50:0.1:0.05. The solution was then degassed via three
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freeze–vacuum–thaw cycles and placed in a 60 °C oil bath for 12 h. p(MPC)–N3 was obtained by
precipitating in excess acetone. See Fig. S3 of SM for FT-IR spectra.
2.3.5. Synthesis of BLA–NCA.
The synthesis of BLA–NCA was carried out using triphosgene. A suspension of BLA (4 g,
18 mmol) in 25 mL of THF was heated to 60 °C under nitrogen atmosphere. A solution of
triphosgene (1.78 g, 6 mmol) dissolved in 25 mL of THF was added dropwise to the stirred
reaction mixture. When the reaction mixture started to become transparent, a stream of dry
nitrogen was bubbled through the solution to remove HCl. After the reaction was complete, the
mixture was precipitated in anhydrous hexane. Further purification of the obtained Bn-ASP-
NCA was accomplished by recrystallization from anhydrous THF/hexane followed by drying
under vacuum at 40 °C. Yield: 87%. IR (KBr, cm−1): 3312 (amide NH), 3000 (aromatic), 1870–
1788 (cyclic anhydride, asym&sym. C=O str.mode), 1728 (ester C=O str.), 1640 (C=O amide I
1
band), 1517 (amide II band) H-NMR (400 MHz, DMF-d6, 298 K): δ (ppm) 8.95 (CH–NH–
C=O), 7.36–7.37 (Ph), 5.13 (CH2–Ph), 4.69 (NH–CH), 2.8–3.0 (O=C–CH2–CH) (see Fig. S4 of
SM).
2.3.6. Synthesis of Alkyne-terminated p(BLA)n (n= 10, 25, 50, 75).
PPA–CAM (0.012 g, 0.05 mmol) and a prescribed amount of BLA–NCA were dissolved in
absolute DMF (4 mL each) in two separate Schlenk flasks and subsequently combined using a
transfer needle under nitrogen. The reaction mixture was stirred for 3 days at room temperature
under nitrogen. After polymerization was complete, the solvent was concentrated, and
precipitated in diethyl ether. The alkyne terminated p(BLA) (alkyne–p(BLA)n) (n= 10, 25, 50,
75) was obtained after vacuum drying. The NMR spectrum of alkyne–p(BLA)50) and GPC traces
of alkyne–p(BLA)n (n = 10, 25, 50, 75) are provided in Fig. S5 of SM.
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2.3.7. Synthesis of p(MPC)50-b-p(AA)n (n = 10, 25, 50, 75).
The click cycloaddition reaction between p(MPC)–N3 and alkyne–p(BLA)n was performed
as follows. For example, p(MPC)50–N3 (0.35 g, 0.025 mmol) and alkyne–p(BLA)50 (0.24 g,
0.025 mmol) were dissolved in a 25 mL Schlenk flask containing dry DMF (15 mL) connected
with a nitrogen inlet and covered with a rubber septum. The mixture was stirred for 30 min and
N,N,N,N′′,N′′-pentamethyldiethylenetriamine (10 µL, 0.08 mmol) was added through a sterile
syringe under nitrogen environment. The mixture was degassed over three freeze-thaw cycles
and added to another Schlenk containing CuBr (0.011 g, 0.08 mmol) via a nitrogen-purged
syringe. The Schlenk flask was placed in an oil bath maintained at 40 °C for 3 days under static
nitrogen pressure. After the completion of the reaction, the solution was passed through a short
silica column to remove copper catalyst, p(MPC)50–b–p(BLA)50 was precipitated by pouring into
excess diethyl ether (see 1H NMR spectrum in Fig. S6(A) of SM ).
For the deprotection of the protective side groups, a round-bottomed flask was charged with
a solution of p(MPC)50–b–p(BLA)50 in trifluoroacetic acid (250 mg, 5 mL). Then, a four-fold
molar excess of a 33 wt% solution of HBr in acetic acid was added, the reaction mixture was
stirred overnight at room temperature, and then precipitated in diethyl ether to obtain the
deprotected block copolymer p(MPC)50–b–p(AA)50. The trithio moiety was subsequently
removed by aminolysis, in presence of hexylamine (2 eq.) and few drops of Na2S2O4 in DMF,
and the block polymer was dialyzed against DI water for 24 h. The lyophilized p(MPC)50–b–
1
p(AA)50 (see H NMR spectrum in Fig. S6(B) of SM) stored at −20 °C. Other copolymers also
were synthesized, purified and stored by similar procedure.
2.4. Fabrication of p(MPC)50–b–p(AA)n vesicles.
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The vesicles of p(MPC)50–b–p(AA)n were prepared by combining a self-assembly derived
nanoprecipitation with membrane dialysis against double distilled water. For example,
p(MPC)50–b–p(AA)50 (1 mg) was dissolved in ethanol (0.5 mL), followed by the dropwise
addition of phosphate buffer (PBS) (0.5 mL, pH 7.4, 50 mM) under stirring. The mixture was
then heated to 50 °C for 2 h under static conditions, and allowed to cool slowly. The turbid
mixture was then dialyzed against DI for 12 h using a dialysis membrane (Spectrapor, MWCO =
2000). The outer phase was replaced at 3-h intervals with fresh water, and finally the vesicles
were lyophilized. TEM observation was performed directly after the deposition of the vesicle
dispersion onto a Formvar carbon-coated copper grid after complete drying.
2.5. Reduction and pH-triggered destabilization of p(MPC)50–b–p(AA)n vesicles.
The size change of the vesicles in response to the addition of 1,4-dithiothreitol (DTT) and
pH change was determined by DLS measurements. The solution was mildly stirred at 37 °C and
degassed in advance by purging nitrogen for 10 min, and then at different time intervals, the size
of the vesicles was measured using a DLS spectrometer. Briefly, a 2 mL solution of p(MPC)50–
b–p(AA)75 vesicles (1 mg/mL) in PBS was adjusted to the required pH, then to monitor the pH
and reduction-responsive disassembly, 10 mmoL of DTT was added and the pH corrected
before measurement.
2.6. Preparation of Dox-loaded p(MPC)50–b–p(AA)n vesicles and in vitro Dox release.
For the fabrication of Dox–loaded p(MPC)50–b–p(AA)75 vesicles, Dox (2 mg) was dissolved
in 0.5 mL of ethanol. The ethanolic solution of p(MPC)50–b–p(AA)75 (1 mg in 0.5 mL) and the
Dox solution was then mixed and stirred for 2 h in the dark. PBS (0.5 mL, pH 10.5, 50 mM) was
added under stirring and the mixture warmed to 50 °C for 2 h under static conditions. The
solution was dialyzed using a membrane (Spectrapor, MWCO=2000) for 12 h to remove free
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drug and then lyophilized in the dark. For quantification of the amount of drug encapsulated,
aliquots of the drug-loaded vesicles were broken up by adding 1 mL of DMSO. The obtained
solution was analyzed using UV–Vis spectroscopy. The characteristic absorbance of Dox (485
nm) was recorded and compared with a standard curve generated from a range of Dox
concentrations in DMSO. The percentages of DLC and DLE were calculated according to the
following equations.
⨯100
⨯100
For reduction and pH-triggered in vitro Dox release studies, Dox–loaded vesicles were
diluted to 1 mg/mL and then the solution was transferred into a dialysis membrane tubing
(MWCO =10000). It was immersed into small beakers containing 20 mL of PBS buffer with
fixed pH value and DTT concentrations to acquire the sink conditions and the solutions were
incubated at 37°C in the dark in a shaking bath. At predetermined time intervals, the buffer
solution was replaced (1 mL each) with fresh solution and the amount of the released drug was
measured by UV–vis spectroscopy measurements. The drug concentration was determined
according to the standard curves for the drug solution at different pH values.
2.7. Cell Viability Assay
In vitro cytotoxicity of blank and Dox-loaded p(MPC)50–b–p(AA)n vesicles was assessed
using NIH 3T3 and 4T1 cell lines. The cells were seeded in 96-well plates at a density of 1x104
and incubated overnight at 37 °C before treatment. The media were then removed and
p(MPC)50–b–p(AA)n vesicle samples at a range of concentrations in OPTI-MEM® were added
to the cells, which were then incubated at 37 °C for 6 h. The optimal media was then aspirated
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and replaced with 100 µL of fresh media and incubated for 24 h at 37 °C. Cell viability profiles
were assessed using the MTS assay. Briefly, 20 µL of MTS reagent was added to each well and
incubated for 4 h; cell viability was determined by measuring the absorbance at 490 nm using a
550 BioRad plate-reader. The cell viability in each well was calculated from the measured values
and presented as relative percentages using non- treated cells as a positive control. Results were
presented as the mean and standard deviation, obtained from five different samples.
2.8. Confocal Laser Scanning Microscopy (CLSM) and Flow Cytometer Analysis
To perform CLSM, 4 T1 cells were seeded in a Lab-Tek® chamber slide at a density of
1×105 cells/well and incubated overnight prior to sample addition. The pH of the culture medium
was regulated with 0.1 M HCl or 0.1 M NaOH to give the required pH. After overnight
incubation, Dox loaded p(MPC)50–b–p(AA)75 vesicles were added to the 4T1 cells and incubated
in the dark for 4 h at 37 °C. The media was then aspirated and cells were washed with PBS three
times, followed by 4% paraformaldehyde treatment to fix the cells. Fluorescence was then
visualized using confocal laser scanning microscope.
The flow cytometer analysis Dox loaded p(MPC)50–b–p(AA)75 vesicles were treated with
4T1 cells, seeded at a density of 1×106 cells/well were incubated overnight. The pH of the
culture medium was regulated with 0.1 M HCl, or 0.1 M NaOH to a required pH prior to the
treatment. After incubation, the medium was then aspirated and the cells were washed three
times with PBS. After PBS wash, cells were collected and analyzed with flow cytometer. An
excitation wavelength at 488 nm and emission wavelength at 522 nm were used to observe the
Dox fluorescence intensity.
3. Results and Discussion
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3.1. Synthesis of azide-terminated p(MPC) via RAFT polymerization
RAFT polymerization is well established as the most promising radical polymerization
technique, owing to its ease of experimental setup and its applicability to a wide range of
monomers. Trithiocarbonate CTAs offer flexibility for the design of functional copolymer
architectures directly or after an appropriate end-group modification. The overall synthetic
procedure of p(MPC)–b–p(AA)n (n = 10, 25, 50, 75) is shown in Scheme 1. We utilized CTA–N3
1
bearing an azide group for the polymerization of MPC. The H, 13C-NMR and FT-IR spectra of
the CTA–N3 are provided in Fig. S1 of SM. The RAFT polymerization of MPC was conducted
in methanol with [MPC]:[CTA]:[AIBN] feed ratios of 50:1:0.3. Polymerization was carried out
over 12 h and the azide-terminated p(MPC) purified and isolated by precipitating in acetone. The
1
number average MW (Mn) of the polymer was estimated using H-NMR end-group analysis by
integrating the signals corresponding to MPC (3.1 ppm) and to aromatic signals from the CTA
portion (7.2 ppm). The calculated Mn was 14,000, which corresponds to about 48 repeating units
of MPC and is close to the numbers obtained from theoretical calculations. The p(MPC)–N3 was
further characterized by FT-IR spectroscopy which revealing a characteristic band at 2100 cm−1
confirming the existence of azide groups in the polymer chain end (Fig. S3 of SM).
3.2. Synthesis of alkyne–p(BLA)n
The synthesis of alkyne–p(BLA) was carried out using PPA–CAM as the initiator. The
initiator structure was confirmed by 1H, 13C-NMR, and FT-IR spectral analyses (Fig. S2 of SM).
Subsequently, the ring-opening polymerization of BLA–NCA was performed in anhydrous DMF
and completed in a controlled manner in 1 to 3 days depending on the degree of polymerization
to be obtained. The full conversion of the NCAs into p(BLA) was monitored and confirmed by
the disappearance of the NCA-associated carbonyl peaks at 1795 and 1730 cm−1 in the FT-IR
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spectra. The alkyne–p(BLA)n (n = 10, 25, 50, 75) polymers were precipitated in ethyl ether twice,
and vacuum-dried. All polymers were characterized by 1H-NMR spectra and the Mn values were
calculated by comparing the peak integral ratios of the methylene group (4.9 ppm) and the
aromatic signal (7.4 ppm) from p(BLA) units. Based on GPC characterization, the Đ values of
1
the block copolymers were found to be in the range of 1.2−1.3 (see Table S1). The H-NMR
spectra and the GPC elution curves of alkyne–p(BLA)n are provided in Fig. S5 of SM.
3.3. Synthesis of p(MPC)50−b−p(AA)n
With the aim of synthesizing and evaluating biocompatible and dual stimulus-responsive
block copolymers, we selected p(MPC) and poly(L-aspartic acid) (p(AA)) as the building blocks.
Importantly, p(MPC) is among the most biocompatible polymers known. To date, several
p(MPC)-based therapeutics including therapeutic proteins [46], anti-cancer drug conjugates [47],
and block copolymer micelles [48, 49] or vesicles [50,51] have been studied. p(AA) is a
biodegradable polypeptide containing free carboxylic acid groups in the chain. These carboxylic
acid groups are protonated at pH below 5.0 but are ionized at pH above 5.0 as the pKa of p(AA)
is 4.88 [51]. Its biodegradability and pH-sensitive properties [52] make p(AA) an ideal molecule
to combine with p(MPC) in the design of an effective and sensitive drug delivery system. To the
best of our knowledge, this is the first report of redox and pH dual-responsive p(MPC)−b−p(AA)
block copolymers developed for the intracellular delivery of Dox.
RAFT has already been successfully employed with copper-catalyzed azide-alkyne
cycloaddition in polymer synthesis by introducing azides and alkyne groups either as end groups
or as monomers [53−55]. Herein, for the synthesis of p(MPC)50−b−p(AA)n, azide-terminated
p(MPC)50 was subjected to click cycloaddition reactions under "strict click conditions" with
alkyne terminated p(BLA)n, to yield p(MPC)–b–p(BLA)n (n = 10, 25, 50, 75). The protective
15

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side-chains of the polymers were removed by treating with HBr/AcOH in trifluoroacetic acid and
subsequent aminolysis to remove the thiocarbonylthio end groups. After precipitation in diethyl
ether, p(MPC)50−b−p(AA)n was further purified by dialysis against DI water and the pure
1
product isolated after lyophilization. The block copolypeptides were characterized by H NMR
spectroscopy (see Fig. S6 of SM). The composition and the MW of the block copolypeptides are
provided in Table 1.
3.4. Fabrication of and characterization of p(MPC)50−b−p(AA)n vesicles
Herein, we demonstrated the fabrication of vesicles by the self-assembly of
p(MPC)50−b−p(AA)n hybrid block copolymers. In order to investigate the self-assembling
behavior, we selected block copolymers with a large number of aspartic acid units (n = 50, 75).
After careful examination of polymer solubility, an ethanol-water solvent combination was
selected for the fabrication of the vesicles. It was identified that, the copolymers with a less
aspartic acid units displayed enhanced solubility in water, and thus prevented the formation of
stable vesicle structures through self-assembly in aqueous media. Self-assembly occurred after
injection of aqueous buffer into the ethanolic polymer solution, a step that exploited the
solubility difference of p(MPC) and p(AA) blocks in water and ethanol. After removal of ethanol
by dialysis, the morphologies of the self-assembled vesicles were characterized. We found that
the amount of aqueous buffer injected determined the nature of the formation of the vesicles. It
was determined that addition of 300 µL of aqueous buffer to 700 µL of polymer in ethanol (1
mg/mL concentration) was the most ideal for the formation of uniform vesicles. Dialysis against
deionized water was conducted to remove unloaded Dox and ethanol, and the Dox loading
content and loading efficiency of the vesicles were determined (Table 2). Dox encapsulated in
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the vesicles of p(MPC)50−b−p(AA)75 were utilized for the, in vitro release, and cellular
internalization studies.
The pH and redox-responsive size change of the p(MPC)50−b−p(AA)75 vesicles was
confirmed by a set of DLS experiments. At physiological conditions (pH 7.4 and 37 °C) a
uniform particle size of 100 nm was observed (Fig. 1(a)). Measurement 4 h later, after adjusting
the pH of the vesicle dispersion to that of the endosomal range (pH 5.5), revealed that the
particle size had significantly increased (Fig. 1(b)) i.e., there was a pH-induced swelling of the
vesicles. Interestingly, in this endosomal pH range, addition of 10 mM DTT caused a further
increase in size when measured after 4 h (~800 nm) and a broad distribution was observed (Fig.
1(c)). This phenomenon was attributed to the synergistic effect of the reductive cleavage of the
SS bond together with the pH-induced swelling of the vesicles. Finally, the morphology of the
p(MPC)50−b−p(AA)n (n= 50, 75) vesicles fabricated at pH 7.4 were visualized by TEM. As
shown in Fig. 2, spherical vesicles of around 100 nm were clearly observed and this was
consistent with the size analysis by DLS.
3.5. Stimuli-responsive Release of Dox from p(MPC)50−b−p(AA) Vesicles
Dox is widely used to treat a broad range of solid malignant tumors. Herein, Dox was used
as a model drug to evaluate the in vitro drug release behavior from loaded p(MPC)50−b−p(AA)75
vesicles. Dox was loaded into the vesicles through a dialysis method; the DLC and DLE were
determined to be 19.8% and 66%, respectively (Table 2). The time-dependent release
performance of Dox under different pH conditions and different DTT concentrations was
investigated in p(MPC)50−b−p(AA)75 vesicles in PBS. Dox release was evaluated by UV-Vis
spectrometry at 480 nm which is the characteristic absorption maximum of Dox in solution. The
release performance was assessed over 72 h; release profiles are shown in Fig. 3. At pH 7.4, Dox
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loaded p(MPC)50−b−p(AA)75 vesicles at different DTT concentrations (5, 10, 15 mmol)
displayed a suppressed Dox release profile compared to the corresponding experiment conducted
at pH 5.5. As shown in Fig. 3(a), it is obvious that only about 15%, 31% and 44% of Dox was
released in 72 h, at 5, 10, and 15 mmol DTT, respectively.
On the other hand, at pH 5.5, the Dox release rate was much faster and within 72 h,
61% ,70%, and 76% of the initial Dox load was released at 5, 10, 15 mmol DTT, respectively
(Figure 3(b)). Importantly, at pH 5.5, Dox release is found to be more effective at all DTT
concentrations when compared with that at pH 7.4 at the corresponding DTT concentrations. The
highest Dox release rate was observed when dual stimuli (pH 5.5 and addition of 15 mmol of
DTT) were applied. The dual-stimuli responsive Dox release phenomenon is related to the
structural disassembly of the p(MPC)50−b−p(AA)75 vesicles. While a lower pH led to swelling of
the Dox-loaded vesicles due to the protonation of the carboxyl groups in p(AA) segments, the
concurrent addition of DTT resulted in vesicle disassembly caused by the cleavage of the SS
linker. These release profiles clearly indicated a desirable pH and reduction sensitivity for
intracellular delivery. In other words, the vesicle could hold a substantial amount of encapsulated
Dox over an extended period during systemic circulation (pH 7.4) but rapid release of the drug
can be triggered by the tumoral acidic and reductive environment.
3.6. Cytotoxicity of p(MPC)50–b–p(AA)n vesicles
Investigation of the effect of blank vesicles on cell viability is important prior to the in vitro
and in vivo Dox internalization studies. The effect of p(MPC)50–b–p(AA)n vesicles on the
viability of NIH 3T3 and 4T1 cell lines was determined using an MTS cell viability assay. The
cell-only group was used as the positive control (P.C.) and Triton X100-treated cells were used
as the negative control (N.C.). The effect of p(MPC)50–b–p(AA)n vesicles on the viability of NIH
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3T3 and 4T1 cell lines was assessed over a wide range (1−100 μg/mL) of polymer
concentrations. Results revealed no significant effect on cell viability profiles upon treatment
with p(MPC)50–b–p(AA)n polymers regardless of the chain length of p(AA). Polymer-treated
NIH 3T3 and 4T1 cells showed high viability (>95%) even at the highest exposure levels (Fig. 4).
From the results of the in vitro studies, it was evident that p(MPC)50–b–p(AA)n polymers had no
acute and intrinsic cytotoxicity against normal (NIH 3T3) or cancer cell lines (4T1) irrespective
of their structural composition
3.7. Cellular uptake and intracellular release of Dox
To examine the anticancer effect of the Dox-encapsulated p(MPC)–b–p(AA) vesicles
against cancer cells, Dox-encapsulated p(MPC)50-b-p(AA)75 vesicles were administered to 4T1
cancer cells over 24 h. The Dox-loaded vesicles underwent disassembly, due to the synergistic
effects of redox- and pH stimulus-responsiveness, and displayed a dose-dependent (0.2−100
µg/mL of Dox concentration) cytotoxicity against the 4T1 cell lines. The enhanced toxicity and
cell death that was observed in the endosomal pH range (pH 5.5) highlighted the increased
intracellular release of Dox from the vesicles at that pH when compared with that occurring at
pH 7.4. Note that, at pH 5.5, the clear stimulus-responsive release of Dox resulted in a dose-
dependent anticancer effect from 6.2 µg/mL Dox upwards (Fig. 5(A)).
Confocal microscopic experiments were performed to visualize the intracellular uptake and
redox- and pH-responsive intracellular release of Dox from the vesicles into 4T1 cancer cells. As
shown in Fig. 5(B), at pH 5.5 (g–i) a stronger fluorescence of Dox was observed when compared
with that observed at pH 7.4 (d–f) compared to the control experiments (a–c). Similar
experiments with free Dox showed low intracellular internalization due to the lack of stimuli
responsiveness (Fig. S7(A)). To obtain confirmation of the observed intracellular internalization,
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cells treated with Dox- loaded p(MPC)50–b–p(AA)75 vesicles and with free Dox were subjected
to quantitative analysis using flow cytometry (FACScan).
The FACScan analysis results of the Dox-loaded p(MPC)50–b–p(AA)75 treated 4T1 cells are
provided in Fig. 5(C). It is also evident that the enhanced fluorescence intensity is observed at
the endosomal pH rather than at the physiological pH. From these results, we concluded that the
vesicles are able to efficiently release Dox via reduction and pH dual stimuli-induced
disassembly, and that they can specifically release Dox into tumor cells. On the other hand, the
FACScan analysis of cells treated only with free Dox showed poor intracellular uptake (Fig.
S7(B)).
4. Conclusions
In this study, we successfully synthesized a series of pH and reduction dual stimuli-
responsive block copolymers p(MPC)50–b–p(AA)n by utilizing RAFT, ring opening
polymerization of amino acid NCAs, and azide−alkyne click cycloaddition reactions. Vesicles
self-assembled from the copolymer have pH and reduction responsiveness as verified by DLS.
The p(MPC)–b–p(AA) copolymer vesicles were effectively internalized into NIH 3T3 and 4T1
cell lines. The viability of both cell lines was >95% at an extensive range of polymer
concentrations (1−100 μg/mL). The Dox-encapsulated vesicles preferentially released Dox in
4T1 tumor cells as confirmed by CLSM and FACScan analysis. The prepared vesicles are
nontoxic, and showed a good loading efficiency of Dox. They could rapidly cross cell
membranes and release Dox into cancer cells by endosomal pH reduction-induced disassembly.
These vesicles fabricated were showing pH and reduction dual-responsiveness, hold great
promise as intracellular anticancer drug delivery systems.
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Acknowledgements
This work was supported by the Basic Science Research Program through the National Research
Foundation of Korea (2015R1D1A1A09057372). The authors also thank to the Bio & Medical
Technology Development Program of the NRF funded by the Korean government, MSIP
(2016M3A9E8942069).
Appendix A. Supplementary material
Supplementary data associated with this article can be found in a separated file.
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Tables
Table 1. Molecular characteristic of p(MPC)50−b−p(AA)n block copolymers synthesized by
azide-alkyne click cycloaddition reaction.
Mn ×10-4(g/mol)
Block copolymer
Yield (%)
Theoretical
1.55
NMR
1.42
1.78
2.31
2.83
p(MPC)50−b−p(AA)10
p(MPC)50−b−p(AA)25
p(MPC)50−b−p(AA)50
p(MPC)50−b−p(AA)75
58
66
62
60
1.91
2.42
2.95
Table 2. The behaviors of blank and Dox loaded vesicles of p(MPC)50-b-p(AA)75 at different
pHs.
Dox-loaded
vesicles
Blank
DLC DLE
vesicles
DH (nm)
110±1.6
Block copolymer
pH
at 20:1 (w/w)a
(%)
(%)
Ð
DH (nm)
Ð
p(MPC)50−b−
7.4
0.24
240±2.1
0.23
19.8 66.0
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p(AA)75
5.5
280±1.8
0.46
320±1.3
0.38
[a] Feed ratio of polymer to Dox. Dox loading experiments were performed using PBS (pH 10.5).
Scheme and Figure Captions
Scheme 1. Synthetic route of p(MPC)50−b−p(AA)n block copolypeptides by the click
cycloaddition reaction of azide-terminated p(MPC) and alkyne-terminated p(BLA).
Fig. 1. Size variation, based on DLS measurements, of p(MPC)50−b−p(AA)75 vesicles in
response to pH and addition of DTT (a) at pH 7.4 and (b) at pH 5.5 after 4 h, and (c) at pH 5.5
and 10 mM DTT after 4 h.
Fig. 2. TEM images of p(MPC)50−b−p(AA)75 vesicles obtained by self-assembly in
ethanol/water solvent mixture: (a) at pH 7.4, (b) at pH 5.5, and (c) at pH 5.5 in the presence of 5
mmoL DTT.
Fig. 3. The time-dependent release of Dox from p(MPC)50−b−p(AA)75 vesicles in phosphate-
buffered saline (PBS) at 37 oC and at different DTT concentrations (a) pH 7.4 and (b) pH 5.5.
Fig. 4. Cell viability measured by the MTS assay of (a) NIH 3T3 and (b) 4T1 cells treated with
p(MPC)50–b–p(AA)n at different concentrations. The cell-only group was used as the positive
control (P.C), and Triton X100 was used as negative control (N.C).
Fig. 5. (A) Dose-dependent antitumor activity of Dox-loaded p(MPC)50–b–p(AA)75 vesicles
against 4T1 cells after 24 h incubation at pH 7.4 and 5.5. (B) Confocal laser scanning
microscopic images of 4T1 cells taken after 4 h incubation at pH 7.4 (d–f) and pH 5.5 (g–i) with
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