Dual-responsive star-shaped polypeptides for drug delivery

Article abstract of DOI:10.1039/c5ra20972b

Core cross-linked star-shaped polypeptides based on poly(l-glutamic acid)-poly(l-phenylalanine-co-l-cystine) copolymer have been successfully synthesized and thoroughly characterized. The star polypeptides can self-assemble to form 50 nm micelles in aqueous medium, which respond rapidly to both pH change within the physiologically relevant pH range and a reduction environment mimicking the intracellular space. Water-soluble doxorubicin hydrochloride and hydrophobic resveratrol are loaded into the star polypeptides micelles through electrostatic and hydrophobic interactions respectively. The drug loading content can be controlled by tuning the composition of the star polypeptides. The in vitro release studies indicate dual sensitivity enabled rapid drug release at pH 5.5 and 10 mM dithiothreitol (DTT), mimicking the intracellular environment. Furthermore, the star polypeptides are biocompatible and interact well with cells in vitro. Confocal fluorescence microscopy and flow cytometry assays show these star polypeptides can be quickly internalized and effectively deliver the drugs into HeLa cells to inhibit cell growth.

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Dual-responsive star-shaped polypeptides for drug delivery
Wenlong Wang, #a Liang Zhang, #a Mengtao Liu,a Yuan Le,*a Shanshan Lva and Jian-Feng Chenab
Received 00th January 20xx,
Accepted 00th January 20xx
Core cross-linked star shaped polypeptides based on poly(L-glutamicacid)-poly(L-phenylalanine-co-L-cystine) copolymer
have been successfully synthesized and thoroughly characterized. The star polypeptides can self-assemble to form 50 nm
micelles in aqueous medium, which respond rapidly to both pH change within the physiologically relevant pH range and a
reduction environment mimicking the intracellular space. Water-soluble doxorubicin hydrochloride and hydrophobic
resveratrol are loaded into the star polypeptides micelles through electrostatic and hydrophobic interaction respectively.
The drug loading content can be controlled by tuning the composition of the star polypeptides. The in vitro release studies
indicate dual sensitivity enabled rapid drug release at pH 5.5 and dithiothreitol (DTT) 10 mM mimic to intracellular
environment. Furthermore, the star polypeptides are biocompatible and interact well with cells in vitro. Confocal
fluorescence microscopy and flow cytometry assay show these star polypepides can be quickly internalized and effectively
deliver the drugs into HeLa cells to inhibit cell growth.
DOI: 10.1039/x0xx00000x
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clinical applications are facing tremendous challenges
including poor in vivo stability, low drug loading levels and
slow drug release.19,20 In comparison with conventional linear
1. Introduction
Biodegradable polymeric micelles formed by amphiphilic block
copolymers have been constituting a growing interest in the
fields of drug delivery,1-3 owing to their excellent performance
in enhancing drug solubility, prolonging drug circulation time
and minimizing side effects.4 Various intelligent polymers in
response to internal or external stimuli, such as temperature,5
pH,6,7 light,8 ultrasound,9 and redox potential,10 have been
used extensively by drug delivery systems. Among the
stimulus-responsive polymers, pH and redox-responsive
polymers appeared to be the most attractive candidate for
targeted and controlled drug delivery because of the
significant differences in the glutathione (GSH) concentration
and pH between different physiological tissue.11,12 The
physiological pH of blood and normal tissues is 7.4, but the
extracellular pH in some tumor tissues is 6.8, and the
intracellular endo/lysosomal pH ranges from 4.0 to 5.5.13,14
Meanwhile, the GSH concentration is 2-20 μM in blood and
normal tissue, but it is around 10 mM in tumor cells. 15 Thus,
these dual-stimuli responsive polymers have unprecedented
control over drug release and delivery to increase efficacy in
vitro and in vivo.16-18
polymers, star polymers with three-dimensional architecture
exhibit unique properties such as enhanced stability,21 high
encapsulation capabilities,22 as well as large number of internal
and peripheral functionalities.23,24 They are expected for
potential clinic application. Polypeptides, which are
poly(amino acid)s linked by peptide bonds, are unique
biodegradable and biocompatible polymers with structures
mimicking natural proteins.25 Meanwhile, polypeptides can
offer transitions in response to external stimuli,26,27 and their
side chains could be incorporated with various functional
moieties for designing intelligent polymer systems.28-30
Therefore, it is interesting to explore the potential of stimul-
responsive star polypeptide for improving drug delivery.
To our knowledge, star polypeptides as dual pH and redox-
responsive micelles have not been reported yet. Moreover,
the reported responsive polymers carry drugs just through
physical interaction or via chemical conjugation despite that
both hydrophilic and hydrophobic drugs can be entrapped into
the polymeric micelles.31-33 In addition, the process of
encapsulation, tunable parameters, and biocompatibility are
not well characterized for the application of drug delivery.
There is great demand to design smart system with enhanced
functionality and precise molecular control.
Although much research has been carried out on polymeric
micelles for stimuli-responsive drug delivery systems, their
a.State Key Laboratory of Organic-Inorganic Composites, Beijing University of
Chemical Technology, Beijing 100029, P.R. China
b.Research Center of the Ministry of Education for High gravity Engineering and
Technology, Beijing University of Chemical Technology, Beijing 100029, PR China
# These authors contributed equally to this work.
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Scheme 1. Synthetic strategy toward star polypeptides PLG-P(LP-co-LC).
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
In this work, we designed a novel core cross-linked star and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI)
polypeptides based on poly(L-glutamic acid)-poly(L-pheny- were purchased from Sigma-Aldrich (St Louis, MO, USA) and
lalanine-co-L-cystine) (PLG-P(LP-co-LC)) for dual responsive used as received. Tetrahydrofuran (THF), dichloromethane
drug delivery, as shown in scheme 1. By taking advantages of (DCM), 1,4-dioxane were obtained from Sinopharm Chemical
the star polypeptides properties, PLG-P(LP-co-LC) copolymers
are expected to undergo spontaneous self-assembling to reagents and solvents were analytical grade and used without
micelles in the aqueous solutions resulting in the PLG outer further purification.
Reagent (Beijing, China) and distilled prior to use. All the other
corona and hydrophobic PLP/PLC inner core. Doxorubicin 2.2 Characterizations
hydrochloride (DOX) was selected as a hydrophilic drug and
NMR spectroscopy was performed on Bruker AV
Ⅲ 400 NMR
resveratrol (RES) was used as a hydrophobic drug. RES (trans-3,
5, 4’-trihydroxy-stilbene), a natural polyphenol compound
found in grapes and berries,34,35 is well-known for its activities
of antioxidant, cardioprotect, anti-inflammatory36 as well as
anti-cancers.37,38 The anionic PLG provides the strong
electrostatic interaction with cationic DOX, and the hydro-
phobic PLP serves as a reservoir for RES. The physiochemical
properties, drug loading and release performances of the star
polypeptides have been investigated. Furthermore, cell
cytotoxicity and internalization behavior of the drug loaded
star polypeptides have also been explored to evaluate the
potential for in vivo drug delivery.
spectrometer using the deuterated solvent as reference and a
sample concentration of ca. 20 mgmL-1. FT-IR spectra was
recorded on Bruker VERTEX 70 instrument using the potassium
bromide (KBr) method. The carbon, hydrogen, nitrogen and
sulfur element contents of star polypeptides were estimated
by Vaio EL cube elemental analysis. Scanning electron
microscopy (SEM) measurement was performed on JEOL JSM-
7800F at an accelerating voltage of 10 KV. A drop of the
micelles solution (0.05 gL-1) was deposited onto a piece of
cover glass and allowed to dry in air at 25
℃ before
measurements. Dynamic laser scattering (DLS) measurements
were performed on Malvern ZS90 with a vertically polarized
He-Ne laser (633 nm) at an angle of 173° and a temperature of
25 ± 0.1
℃. The initial PBLG or PBLG-P(LP-co-LC) concen-
2. Experimental
trations of 10 mgmL-1 in DMF were used then serial dilutions
were performed until stable spectra were obtained. All sample
solutions were filtered using 0.45 μm filters. Molecular weight
and molecular weight distributions were determined using
Agilent 2600 Series Gel penetration chromatograph (GPC)
2.1 Materials
L-glutamic acid, L-phenylalanine, L-cystine, dithiothreitol (DTT),
doxorubicin hydrochloride (DOX) and resveratrol (RES) were
purchased from Aladdin Industrial Corporation(Shanghai,
China). Benzyl-L-glutamate (BLG) and N-Benzyloxycarbonyl -L-
cystine (Z-L-cystine) were synthesized according to the
literature procedure.39,40 Hexamethyldisilazane (HMDS)
(99.9%), anhydrous N,N-dimethylformamide (DMF), 3-(4,5-
instrument at 25
℃ using DMF with 0.5 M LiBr as the eluent at
a flow rate of 1 mL min-1. The standard curve was determined
using a series of narrow distribution polystyrene standard
samples.
2.3 Synthesis of BLG-NCA, Phe-NCA and Cys-NCA
2 | J. Name., 2012, 00, 1-3
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The amino acids were converted to their NCA derivatives using water under stirring for 24 h. To remove the solvents, the
either triphosgene or thionyl chloride, respectively. Benzyl-L- solution was dialyzed against deionized water for 72 h.
glutamate (1.8 g, 7.6 mmol) was dissolved in 30 mL THF under
nitrogen. Triphosgene (0.9 g, 2.9 mmol) was added and the analyzing the size and zeta potential of the micelles solution at
mixture was stirred at 40 until the cloudy solution turned different pH values using DLS. Furthermore, the disassembly of
The pH-responsibility of PLG-P(LP-co-LC) was examined by
℃
clear. The solvent was removed in vacuo and the resulting micelles in response to redox stimuli was examined by DLS and
residue was purified by recrystallization with ethyl acetate and 1H NMR spectroscopy. DTT was dissolved in degassed micelles
n-hexane to afford γ-Benzyl-L-glutamate-N-carboxyanhydride solution (10 mM DTT) under nitrogen. The obtained solution
1
(BLG-NCA) (yield: 95.1%). H NMR (400 MHz, CDCl3, δH, ppm): was stirred at 37
℃, and the size of PLG-P(LP-co-LC) was
2.21 (m, 2H, -CH2-), 2.60 (dd, 2H, -CH2COO), 4.45 (t, 1H, -CHN-), determined by DLS at different time intervals. After 24 h, the
5.15 (s, 2H, -CH2O), 7.35 (m, 5H, ArH-), 6.28 (s, 1H, -NH
-
). L- resulting solution was dialyzed and lyophilized for 1H NMR
phenylalanine N-carboxyanhydride (Phe-NCA) was synthesized analysis.
following the same procedure as the synthesis of the BLG-NCA 2.6 Preparation of drug loaded micelles
(yield: 91.5%). 1H NMR (400 MHz, CDCl3, δH, ppm): 3.15 (m, 2H,
The DOX-loaded micelles were prepared according to the
following method. In brief, PLG-P(LP-co-LC) (50 mg) was
dissolved in distilled water and DOX (10 mg) dissolved in
DMSO was added dropwise. After stirring overnight in the
dark, the organic solvent and free DOX were removed by
-CH2-), 4.53 (dd, 2H, -CHN-), 5.78 (s, 1H, -NH
ArH-).
-), 7.30 (m, 5H,
Z-L-cystine (2.0 g, 3.9 mmol) was dissolved in 20 mL 1,4-
dioxane. Thionyl chloride (1.2 mL, 16.5 mmol) was added and
the mixture was reacted at 55
℃ for 3h. After removal of the
dialysis using
a dialysis bag (MWCO=3500 Da) against
solvent in vacuo, the resulting residue was precipitated in DCM
to obtain L-cystine N-carboxyanhydride (Cys-NCA) (yield:
1
92.3%). H NMR (400 MHz, DMSO-d6, δH, ppm) 3.20 (m, 4H, -
deionized water for 24 h, and then freeze-dried to obtain the
DOX-loaded micelles.
The RES-loaded micelles were prepared by a dialysis method.
Briefly, 50 mg of PLG-P(LP-co-LC) and 5 mg of RES were
dissolved in DMSO, and the solution was added dropwise to
SCH2CH-), 4.77 (dd, 2H, -CHN-), 9.25 (s, 2H, -NH-).
2.4 Synthesis of star polypeptides PLG-P(LP-co-LC)
The star polypeptides PLG-P(LP-co-LC) were synthesized the deionized water under stirring. The mixture was
through one-step ring-opening polymerization of BLG-NCA, maintained at room temperature under stirring for 12 h. The
Cys-NCA and Phe-NCA in DMF using HMDS as the initiator. In solution was dialyzed against excess deionized water with a
brief, BLG-NCA (1.0 g, 3.3 mmol) and HMDS (17 μL, 82 μmol) dialysis bag (MWCO=3500 Da) for 24 h, and then filtered
was dissolved in anhydrous DMF (10 mL) in a glove box. The through a 0.45 μm pore-sized microporous membrane. The
reaction mixture was stirred at room temperature until the RES-loaded micelles were obtained after lyophilization.
NCA’s anhydride peak at 1786 cm-1 disappeared, as DOX loaded micelles were determined by UV absorption at
determined by FT-IR. After diluting the concentration of 480 nm and RES loaded micelles were detected at 306 nm. The
poly(γ-benzyl-L-glutamate) (PBLG) to 1 mgmL-1 using DMF drug loading content (DLC, wt%) and drug loading efficiency
(containing 0.1 M LiBr) for measuring the molecular weight of (DLE, wt%) were calculated according to the following
PBLG. Then, Phe-NCA (0.4 g, 2.3 mmol) and Cys-NCA (0.3 g, 1.0 formulas
mmol) were added to the remaining PBLG solution, and the
reaction was maintained for another 10 h. The polymer
solution was precipitated into excess amount of methanol to
afford the resulting star polypeptides PBLG-P(LP-co- LC) (yield:
86.5%). 1H NMR (400 MHz, TFA-d6, δH, ppm): 2.21 (d, 2H, -


amount of loaded drug
DLC=
DLE=
× 100%




amount of drug -loaded micelles


amount of loaded drug
amount of feeding drug
× 100%




CHCH2-), 2.48 (s, 2H, -CH2COOH), 4.80 (s, 2H, CH2C6H5), 5.33 (s,
H, -CHN), 7.78 (m, 5H, ArH-).
2.7 In vitro drug release
Subsequently, star polypeptides PBLG-P(LP-co-LC) (2.0 g) were
dissolved in TFA (10 mL) and 4 mL HBr (33% in acetic acid) was
added. After stirring for 1 h at room temperature, the mixture
was precipitated into excess amount of ice diethyl ether. After
dried under vacuum, the crude product was purified by dialysis
against deionized water (MWCO=3500 Da), and lyophilized to
afford water soluble star polypeptides PLG-P(LP-co-LC),
The release profiles of drugs from micelles were investigated
in PBS at different pH values (7.4, 6.8 and 5.5) or different
concentrations of DTT (0 mM, 5 mM and 10 mM) by the
dialysis method. The weighed freeze-dried drug loaded
micelles were suspended in 5 mL of the release medium and
transferred into a dialysis bag (MWCO
experiment was initiated by placing the dialysis bag into
release medium at 37 with continuous shaking at 100 rpm.
=3500 Da). The release
1
yielding a white solid (yield: 87.2%). H NMR (400 MHz, D2O,
δH, ppm): 1.92 (d, 2H, -CHCH2-), 2.23 (s, 2H, -CH2COOH), 4.26
(s, H, -CHN-), 7.26 (m, 5H, ArH-).
℃
At predetermined intervals, 5 mL of the incubated solution
was withdrawn and replaced with equal volume of fresh
release medium. The amounts of DOX and RES were
determined using UV-Vis spectrometer (SHIMADZU UV 2600)
2.5 Stimuli Responsive of star polypeptides
The blank micelles were prepared by dialysis method. The star
polypeptides PLG-P(LP-co-LC) were dissolved in dimethyl
sulfoxide (DMSO), and the solution was added to deionized
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at 480 nm and 306 nm, respectively. All measurements were
conducted in triplicate.
3. Results and discussion
3.1 Synthesis and characterization of star polypeptides PLG-P(LP-
co-LC)
2.8 Cell cultures
HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum (FBS),
Supplemented with 50U mL-1 penicillin and 50U mL-1
The star polypeptides PLG-P(LP-co-LC) were synthesized throu-
gh core cross-linked strategy using sequential ring opening
polymerization of BLG-NCA, Phe-NCA and Cys-NCA using
HMDS as initiator, followed by deprotecting the groups in
HBr/acetic acid. The structures of NCA monomers were
Identified by FT-IR and 1H NMR. Fig. 1S showed the FT-IR
spectra of NCA monomers with various amino acid. The peaks
centered at 1750 and 1850 cm-1 were assigned to
characteristic peak of carbonyl from anhydride. As shown in
Fig. 2SA, the resonances at 7.35 ppm (peak a) and 6.28 ppm
(peak d) were ascribed to the Ar of BLG and NH of NCA. In Fig.
2SB, the peaks at 4.53 ppm (peak c) and 5.78 ppm (peak b)
were assigned to the CH and NH of anhydride. Peaks a
indicated the presence of benzene in Phe-NCA. Fig. 2SC
showed that the peaks at 9.25 ppm and 3.20 ppm were
ascribed to NH and CH2 of Cys-NCA. The 1H NMR and FT-IR
spectroscopy confirmed the successful synthesis of NCA
monomers.
streptomycin, and incubated at 37
℃ in 5% CO2 atmosphere.
2.9 Cell viability assays
The in vitro cytotoxicities of blank micelles, free drugs, and
drug loaded micelles were assessed with MTT assays. HeLa
cells were seeded in 96-well plates at ~4000 cells per well in
100 μL of DMEM containing 10% fetal bovine serum,
supplemented with 50U mL-1 penicillin and 50U mL-1 strepto-
mycin, and incubated at 37
℃ in 5% CO2 atmosphere for 24 h.
The culture mediums were replaced with 200 μL fresh
mediums containing different concentrations of star
polypeptides (0~
500 μgmL-1). The absorbency of the solution
was measured on Multiskan MK3 microplate reader at 490 nm.
The cell viability (%) was calculated by (Asample/Acontrol) × 100,
where Asample and Acontrol denoted as the absorbencies of the
sample and control wells, respectively. Data are presented as
means ± standard deviation (n = 3).
Then, the polymerization initiated by HMDS was monitored via
FT-IR intensity of NCA’s anhydride peak at 1787 cm-1,41,42 the
disappearance of NCA’s anhydride peak confirmed the
complete consumption of the monomers. Upon addition of
Phe-NCA and cross-linker Cys-NCA, the weight average molar
mass(Mw) of polypeptides increased from 7.5 kDa to 932.4
kDa (Fig. 1A), and the size measured by DLS significantly
increased from 8.5 nm to 101.3 nm (Fig. 1B). The 1H NMR
spectrum of PBLG-P(LP-co-LC) (Fig. 2B) showed the character-
istic signals (peak a) in BLG units, but the signals of the cross-
linked core component were not visible due to the
2.10 Intracellular drug release
The cellular uptake and intracellular release behaviors of DOX
loaded micelles were determined by confocal laser scanning
microscopy (CLSM) toward HeLa cells. The cells were seeded
on coverslips in 6-well plates with a density of ~40 000 cells
per well in 2 mL of DMEM and cultured for 24 h, and the cells
were incubated with DOX loaded micelles at a concentration of
20 μgmL-1 in complete DMEM. After 4 h, 12 h and 24 h
incubation, the culture medium was removed and cells were
washed three times with PBS. Then, the cells were fixed with
4% paraformaldehyde at room temperature, and the cell
nuclei were stained with 4’, 6-diamidino-2-phenylindole
(DAPI). The treated cells were visualized under a laser scanning
confocal microscope (Leica TCS SP5).
The cellular uptake of micelles was also analyzed quantitatively
using flow cytometry. HeLa cells were seeded at a density of
2×104 cells per well in 6-well plates and incubated for 24 h.
After incubating with DOX loaded micelles (at a concentration
of 20 μgmL-1) for 4, 12 and 24 h, the cells were washed three
times with PBS, harvested and subsequently resuspended in
0.5 mL PBS for flow cytometry analysis (FC500, Beckman
Coulter, USA). The instrument was calibrated with non-treated
cells (negative control) to identify viable cells, and the cells
were determined from a fluorescence scan performed with
1×104 cells using the FL1-H channel.
2.11 Statistical analysis
All the values were presented as mean ± standard deviation
(SD) of at least three independent measurement. Statistical
significance was tested by one-way ANOVA followed by a
Student’s t-test for multiple comparison tests. Difference
characterized by *P < 0.05 were considered statistically signify-
cant. All statistical analysis was performed using SPSS, version
22.
Fig. 1 GPC RI chromatograms (A) and size (B) of PBLG and PBLG-P(LP-co-LC)
in DMF.
4 | J. Name., 2012, 00, 1-3
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composition ratio of the monomeric repeating units in PLG,
PLP and PLC blocks was 40:24:10 (calculated by elemental
analysis).
3.2 Stimuli Responsive of Star polypeptides
The pH and redox-responsive behaviors of the star
polypeptides PLG-P(LP- co-LC) were investigated by DLS and 1H
NMR, as shown in Fig. 3 and Fig. 4. The zeta potentials of PLG-
P(LP-co-LC) significantly increased as the pH decreased, and
the carboxyl groups in PLG were in a fully deionized state
below pH 5.0, resulting in significant agglomeration.45 Similar
pH-dependent agglomeration was also proved by the size (Fig.
3), which dramatically increased to 1306 nm with the decrease
of pH from 8 to 3. It is well known that the disulfide linkages
are stable under normal physiological conditions but respond
to reductive conditions (e.g. GSH, DTT) via reversible cleavage
into free thiols. The star polypeptides PLG-P(LP- co-LC) could
preserve their core-shell structures without DTT. In the
presence of DTT (10mM), significant change of size was
observed, wherein the size gradually increased from 80 to 140
nm in 5 h and over 290 nm in 24 h (Fig. 4A). These results
suggest that the disulfide linkages of PLG-P(LP-co-LC) were
cleaved by 10 mM DTT so that the core structure of star
polypetides micelles dissociated that lead to the size increase.
Meanwhile, the characteristic signals corresponding to the
core segments were also observed in 1H NMR spectra after
cleavage, including signals at 7.2 ppm (C6H5- in PLP) and 2.9-
3.2 ppm (-CH2- in PLC) (Fig. 4B).
3.3 characterization of drug loaded micelles
The star polypeptides PLG-P(LP-co-LC) with hydrophilic PLG
arms and hydrophobic PLP core spontaneously formed core-
shell micelles in aqueous solutions. The morphologies of the
micelles were studied by SEM (Fig. 5A), which showed that
micelles were spherical shaped with an average diameter of
around 50 nm. In contrast, the size measured by DLS were 80
nm (Fig. 5D). The smaller values from SEM observations should
be due to the dehydration of the micelles during the SEM
sample preparation process. Furthermore, DOX and RES were
loaded into the micelles by nanoprecipitation technique.
Compared to blank micelles, the size of drug loaded micelles
Fig. 2 FT-IR spectra (A) of star polypeptides PLG-P(LP-co-LC) (a) and PBLG–P
(LP-co-LC) (b); 1H NMR of PBLG-P(LP-co-LC) (B, TFA-d6) and PLG-P(LP-co-LC)
(C, D2O).
reduced segmental mobility.43,44 Furthermore, PLG-P(LP-co-LC)
was prepared by removing the benzylox-ycarbonyl groups of
PBLG-P(LP-co-LC). The disappearrance of the ester C=O
stretching peak at 1725 cm-1 (Fig. 2A) and the benzyl group
signal at 7.36 ppm (Fig. 2C) indicated the complete removing
of the protective group in the polypeptides. The final molar
Fig. 3 Size and zeta potential of star polypeptides PLG-P(LP-co-LC) at
different pH.
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Table 1 Characterizations of the drug loaded micelles
DLC (%)
DLE (%)
Star Polypeptides
Feed ratioa
Resultant ratiob
DOX
RES
DOX
RES
PLG-P(LP-co-LC)-1
PLG-P(LP-co-LC)-2
PLG-P(LP-co-LC)-3
1.0 / 0.7
1.0 / 1.0
1.0 / 1.2
1.0 / 0.6
1.0 / 0.8
1.0 / 1.0
15.8
13.2
7.8
5.8
6.9
89.7
81.6
65.3
28.5
34.3
50.5
10.1
aFeed molar ratio of BLG NCA/Phe NCA.
bResultant molar ratio of PLG /PLP calculated by elemental analysis.
Fig. 5 SEM micrographs of blank micelles (A), DOX loaded micelles (B)
and RES loaded micelles (C); Size and zeta potentials (D) of blank
micelles (a), DOX loaded micelles (b) and RES loaded micelles (c).
Fig. 4 Size (A) and 1H NMR spectra (B, D2O) of star polypeptides PLG-P(LP-co-
LC) response to 10 mM DTT.
with PLP, and the zeta potentials of RES-loaded micelles
displayed a slight change. In order to prove the hypothesis, we
adjusted compositions of the polypeptides and investigated
the DLC and DLE of both drugs (Table 1). With the ratio of
hydrophilic PLG decreasing, the DLC of DOX reduced from
15.8% to 7.8% and DLE decreased to 65.3%, which was
attributed to the weakened electrostatic attraction between
DOX and PLG. In contrast, with the ratio of PLP increasing,
hydrophobic interaction between RES and the core was
enhanced, leading to an increase in the DLC and DLE of RES to
10.1%and 50.5%, respectively.49
increased and maintained a narrow distribution as shown in
Fig. 5D, which confirmed drugs being effectively entrapped
into micelles. Meanwhile, the morphologies of the drug loaded
micelles were measured by SEM measurement. SEM images
revealed that all micelles possessed uniformly spherical
morphologies.
Then, drug loading capacity (DLC) and efficiency (DLE) were
measured and shown in Table 1. The DLC and DLE values of
DOX were higher than that of RES owing to different
interactions between drugs and micelles. After DOX loading,
the zeta potentials of micelles changed from -50.1 mV to -19.3
mV due to the carboxyl groups neutralized, suggesting that the
positively charged DOX was captured by the carboxyl groups of
PLG through electrostatic attraction.47,48 However, RES was
loaded into micelles through aromatic stacking interaction
3.4 Release behavior of drug loaded micelles
The DOX and RES release behaviors of micelles were assessed
using a dialysis method at 37
℃ in phosphate buffered saline
(PBS) at different pH values (7.4, 6.8 and 5.5) and different
concentrations of DTT (0 mM, 5 mM and 10 mM). As shown in
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Fig. 6, the release of encapsulated drugs through a change in
pH have been exploited. The release of DOX was greatly
influenced by the environmental acidity, about 22% and 71%
of DOX were released at pH 7.4 and pH 5.5 after 72 h (Fig. 6A).
However, the release of RES performed an opposite trend (Fig.
6B), with only 20% of RES released at pH 5.5 after 72 h. This pH
dependent release behavior was attributed to the deionized
carboxyl groups, which resulted in weakened electrostatic
interaction and strengthened hydrophobic interaction.
Meanwhile, the drug release behaviors under reductive
conditions were also investigated. RES release was accelerated
in PBS with 10 mM DTT (Fig. 6D), about 80% of RES was
released in 72 h due to the swelling of the micelles caused by
the cleavage of the disulfide bonds. In comparison, the stable
electrostatic interaction between the drug and the micelles
resulted in only a slight increase in DOX release (Fig. 6C).
Furthermore, both drugs rapidly released under simulated
endosomal condition (PBS buffer at pH 5.5 with 10 mM DTT),
especially in the first 10 h. Within 72 h, over 85% DOX and 72%
RES were released. These results indicated that the pH and
redox-sensitive stars could effectively release loaded drugs in
endosomal environments.
3.5 In vitro cytotoxicity studies
The cytotoxicity of the star polypeptides PLG-P(LP-co-LC) was
evaluated using MTT assay. HeLa cell lines were utilized. As
shown in Fig.7, the viabilities of HeLa were ~
100% at 30 μgmL-1
for 24 h, gradually decreased with increased concentration of
polymer and incubated time. However, the viabilities of HeLa
were above 80% at all test concentrations up to 500 μgmL-1,
revealing remarkable safety and biocompatibility of the stars
PLG-P(LP-co-LC).
To verify the effect of drug-loaded system, the proliferation
inhibition effects of free drugs and drug loaded micelles
against HeLa cells were tested using MTT assay. The cell
viability histograms were shown in Fig. 8. After 24 h, 48 h, and
72 h incubation, all free drugs and drug loaded micelles
showed dose and time-dependent cell proliferation inhibition
behavior. The cell viabilities incubated with free DOX and DOX-
loaded micelles had similar change trends. With the increase
of drug concentration from 0.6 to 10 μgmL-1, cell viabilities
decreased from 80% to 30% for 24 h. As the extension of
cultivation time, the drugs were taken more into cells thus
result in the decrease of cell viabilities. For RES, cell toxicity
was lower than DOX. The IC50 values of free drugs and drug-
loaded micelles were summarized in Table 2. The IC50 values of
DOX loaded micelles were 2.05, 1.26 and 0.84 μgmL-1 for 24,
48 and 72 h, respectively, which were similar to that of free
DOX. The IC50 values of RES loaded micelles were less than that
of free RES.
Fig. 6 In vitro DOX (A, C) and RES (B, D) release in PBS at 37 ℃.
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Table 2 IC50 (μgmL-1) of free drug and the drug loaded micelles.
Free
DOX
DOX loaded
micelles
RES loaded
micelles
Free RES
2.20
1.32
0.89
24h
48h
72h
2.05
1.26
0.84
33.34
27.31
20.08
22.81
17.13
11.40
Fig. 8 Cell viabilities of HeLa cells incubated with free DOX (A), free RES (B),
DOX-loaded micelles (C), RES-loaded micelles (D) for 24 h, 48 h and 72 h (n =
3, mean ± SD) (*p<0.05, **p<0.01).
Fig. 7 Cell viabilities of HeLa incubated with PLG-P(LP- co-LC) for 24 h, 48 h
and 72 h (n = 3, mean ± SD).
3.6 Cellular uptake behavior of DOX loaded micelles
The cellular uptake and intracellular release behavior of drug
loaded micelles in HeLa cells were investigated by CLSM and
flow cytometry. The cellular nuclei were stained with DAPI
(blue). DOX (red) was loaded in PLG-P(LP-co-LC) for subcellular
observation. As shown in Fig. 9A, DOX fluorescence could be
observed in the cell cytoplasm and distributed widely in the
cytoplasm after 4 h incubation, indicating that the drug loaded
micelles could be successfully internalized by cancer cells via
endocytosis. When the incubation period increased to 12 h
and 24 h, the cell uptake of drug loaded micelles was
enhanced and the fluorescence became even stronger.
Meanwhile, the DOX was mainly located in cytoplasm and
partly entered into nuclei for DOX loaded treated cells (Fig. 9B
and Fig.9C), suggesting that the DOX had been released and
escaped from the endo/lysosomes to the nuclei.
Fig. 9 CLSM images of HeLa cells incubated with DOX loaded micelles after 4
h, 12 h and 24 h.
control cells. Moreover, the mean fluorescence intensity (MFI)
of HeLa cells incubated DOX-loaded micelles for 4 h was 10.8,
which increased to 21.6 after 24 h incubation, indicating a
time-dependent characteristic. These results demonstrated
that the stars can efficiently deliver and release drug into
tumor cells. The reason might be attributed to different cell
uptake pathways of free drugs and drug loaded micelles, and
the controlled release manner of drug-loaded micelles.50
The cellular uptake of DOX loaded micelles was further
quantitative evaluated by flow cytometry, and HeLa cells were
penetrated with the same process as fluorescence microscopy.
As displayed in Fig. 10, the histograms of the pretreated cells
incubated with drug loaded micelles shift clearly to the
direction of high fluorescence intensity compared with the
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Excellent Talents in University of China (NCET-12-0760).
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