Novel shell-cross-linked micelles with detachable PEG corona for glutathione-mediated intracellular drug delivery

Article abstract of DOI:10.1039/c2sm27028e

A series of novel disulfide-containing triblock copolymers, poly(ethylene glycol)-b-poly(l-lysine)-b-poly(rac-leucine) (PEG-SS-PLys-PLeu), were prepared. In an aqueous solution, the copolymers could self-assemble to form core-shell-corona micelles with a disulfide-linked detachable PEG corona, since the PLys middle shell with primary amine groups was linked by a disulfide-containing cross-linker. The morphology and stability of self-assembled micelles were characterized by TEM, DLS and SEM. In the intracellular environment, the micelles underwent destruction of the cross-linked shell with detachment of the PEG corona due to the cleavage of disulfide bonds, followed by the collapse of micelles. The in vitro drug release in response to GSH was further studied. Interestingly, it was found that the micelles not only exhibited reduced drug loss in extracellular environments, but also drastically accelerated drug release at the cytoplasmic GSH level, leading to enhanced growth inhibition of HeLa cells. The glutathione-responsive micelles might have great potential in intracellular drug delivery.

Full text of DOI:10.1039/c2sm27028e

Soft Matter
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PAPER
Novel shell-cross-linked micelles with detachable PEG
corona for glutathione-mediated intracellular drug
delivery†
Cite this: Soft Matter, 2013, 9, 692
Kang Wang, Yun Liu, Wen-Jie Yi, Cao Li, Yong-Yong Li, Ren-Xi Zhuo
and Xian-Zheng Zhang*
A series of novel disulfide-containing triblock copolymers, poly(ethylene glycol)-b-poly(L-lysine)-b-poly(rac-
leucine) (PEG-SS-PLys-PLeu), were prepared. In an aqueous solution, the copolymers could self-assemble to
form core–shell-corona micelles with a disulfide-linked detachable PEG corona, since the PLys middle shell
with primary amine groups was linked by a disulfide-containing cross-linker. The morphology and stability
of self-assembled micelles were characterized by TEM, DLS and SEM. In the intracellular environment, the
micelles underwent destruction of the cross-linked shell with detachment of the PEG corona due to the
cleavage of disulfide bonds, followed by the collapse of micelles. The in vitro drug release in response to
GSH was further studied. Interestingly, it was found that the micelles not only exhibited reduced drug
loss in extracellular environments, but also drastically accelerated drug release at the cytoplasmic GSH
level, leading to enhanced growth inhibition of HeLa cells. The glutathione-responsive micelles might
have great potential in intracellular drug delivery.
Received 2nd September 2012
Accepted 17th October 2012
DOI: 10.1039/c2sm27028e
www.rsc.org/softmatter
18
copolymers. The micelles were composed of PEG outer
coronas, cross-linkable middle shells and hydrophobic cores.
Introduction
Over the past decades, PEG-coated micelles have been consid- The cross-linked shell can hold the nanostructure of the
1
,2
3,4
ered as effective vehicles for drug and gene delivery. The
PEG surface layers of micelles could reduce uptake by macro-
phages of the mononuclear phagocyte system (MPS) and protect
the inner cores from protein opsonization in blood, leading to
assemblies in blood and avoid burst release in the initial stage.
However, this method could not accelerate drug release in the
19,20
cytoplasm.
On the other hand, modication of PEG-shedding was also
5
–7
21–23
prolonged blood circulation time of micelles. However, PEG- excessively exploited in PEG-coated micelles.
For example,
coated micelles still have limitations in practical applications. Kataoka et al. prepared PEG-b-poly(aspartamide) copolymer
Before reaching the pathological site, the micelles suffer from based polyplex micelles, which could detach their PEG shells in
24
poor structural stability and the probable disassembly under the intracellular reducing environment, for gene delivery. The
8
–11
dilute conditions, resulting in undesired drug loss.
Aer the
micelles exhibited 1–3 orders of magnitude higher gene trans-
fection efficiency with a more rapid onset of gene expression.
The removal of the PEG shell aer arrival at the target site would
micelles reach the pathological site, the PEG shielding layer
would act as a diffusion barrier, which delays the release of the
encapsulated payload. Inefficient drug release in the cytoplasm cause disequilibration of the hydrophilic/hydrophobic balance,
may cause undesirable effects on the therapeutic efficacy, even resulting in destruction of the physical structure of the micelles,
12–14
inducing some side effects such as drug resistance.
which would drastically improve the release of therapeutic
To solve these problems, various approaches have been agents. Furthermore, the sudden disassembly is helpful to
1
5,16
25
exploited.
Wooley et al. developed an effective strategy to promote the endosomal escape.
17
prepare micelles with crosslinked shells. This strategy has
been considered as a powerful approach to improve the struc-
tural stability of micelles. Armes et al. reported the preparation
Recently, stimuli-responsive drug delivery systems with
disulde linkages that are cleavable in response to the intra-
cellular redox environment have been widely exploited.
26–29
The
of micelles with cross-linked shells from ABC triblock thiol–disulde exchange reaction plays an important role in the
metabolic activity of living cells. Glutathione (g-glutamyl-cys-
teinyl-glycine; GSH) is the most abundant thiol-containing
Key Laboratory of Biomedical Polymers of Ministry of Education & Department of
peptide in animal cells. In the cellular exterior, the concentra-
Chemistry, Wuhan University, Wuhan 430072, P. R. China. E-mail: xz-zhang@whu.
tion of GSH is very low (approximately 2–20 mM). In contrast, the
edu.cn; Fax: +86 27 6875 4509; Tel: +86 27 6875 5993
concentration of GSH is much higher (0.5–10 mM) in the cyto-
plasm. Furthermore, the concentration of GSH in tumor tissues
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c2sm27028e
1
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30
is at least 4-fold higher than that in normal tissues. Due to the
difference in the GSH concentrations, the delivery vehicles with
disulde linkages could rapidly release the therapeutic agents
intracellularly.
As far as we know, to date, PEG-coated micelles which satisfy
both the requirements of enhanced physical stability and
improved intracellular drug release property have not been
reported.
In this work, we synthesized a novel triblock copolymer
poly(ethylene glycol)-b-poly(L-lysine)-b-poly(rac-leucine) (PEG-
SS-PLys-PLeu), in which the PEG segment was attached with the
PLys block via a disulde linkage to achieve the redox-sensitive
PEG shedding. As shown in Scheme 1, in an aqueous solution,
the copolymers could self-assemble into micelles with the PEG
corona, PLys shell and PLeu core. The shell could be cross-
linked using a disulde-containing cross-linker to enhance
structural stability. In the presence of GSH, the micelles
undergo destruction of the cross-linked shell and removal of the
PEG corona due to the cleavage of disulde bonds, followed by
the collapse of the micelles. The in vitro drug release from the
micelles showed that the micelles hold great promise for
intracellular drug delivery.
Scheme 2 Synthesis of the PEG-SS-PLys-PLeu copolymer.
Synthesis of 3-benzyloxycarbonyl-L-lysine N-carboxyanhydride
Lys(Z)-NCA) and leucine N-carboxyanhydride (Leu-NCA)
Lys(Z)-NCA and Leu-NCA were synthesized according to a liter-
(
31
ature procedure. The detailed procedure for synthesizing
Lys(Z)-NCA is as follows. A THF solution (30 mL) of triphosgene
Experimental part
Materials
(2.5 g, 8.4 mM) was added dropwise to a dry THF suspension
0
Bromoacetic acid, cystamine dihydrochloride, 3,3 -dithiodipro- (70 mL) of Lys(Z) (5.6 g, 20 mM) and the mixture was stirred at
ꢀ
pionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide 50 C under N
2
atmosphere. The reaction was stopped when the
hydrochloride (EDC$HCl), and poly(ethylene glycol) methyl suspension turned to a clear solution. Crude crystals of Lys(Z)-
ether (mPEG) (M
5000) were purchased from Sigma Chemical NCA were obtained by precipitation with an excess of dried
w
Co. and used without further purication. Camptothecin (CPT) n-hexane and recrystallized from dried THF/n-hexane twice and
was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. dried in vacuum. The detailed procedure for synthesizing Leu-
(
China) and used as received. 3-Benzyloxycarbonyl-L-lysine, NCA is as follows. A THF solution (30 mL) of triphosgene (2.5 g,
D-leucine and L-leucine were purchased from GL Biochem 8.4 mM) was added dropwise to a dry THF suspension (70 mL)
0
(
Shanghai) Ltd. Triphosgene was recrystallized before use. N,N - of D-leucine (1.3 g, 10 mM) and L-leucine (1.3 g, 10 mM). The
ꢀ
Dimethylformamide (DMF) and tetrahydrofuran (THF) were of mixture was stirred at 50 C under N
2
atmosphere. The reaction
analytical grade and were distilled prior to use. All other was stopped when the suspension turned to a clear solution.
solvents were of analytical grade and were used without further Crude crystals of Leu-NCA were obtained by precipitation
purication.
with an excess of dried n-hexane and recrystallized from dried
THF/n-hexane twice and dried in vacuum.
Synthesis of PEG-SS-PLys(Z)
PEG-SS-PLys(Z) copolymers were prepared by ring opening
polymerization of Lys(Z)-NCA initiated by PEG-SS-NH
The feed weight ratio of PEG-SS-NH /Lys(Z)-NCA was 2 : 1. PEG-
SS-NH and Lys(Z)-NCA were dissolved in 10 mL anhydrous
DMF and stirred at 25 C under N
2
in DMF.
2
2
ꢀ
2
atmosphere for 72 h; then
the reaction mixture was poured into a large excess of diethyl
ether to precipitate PEG-SS-PLys(Z) twice. The resultant copoly-
mers were dried under vacuum.
Synthesis of PEG-SS-PLys(Z)-PLeu
Leu-NCA and PEG-SS-PLys(Z) were dissolved in 10 mL anhy-
ꢀ
drous DMF and stirred at 25 C under N atmosphere for 72 h;
2
then the resultant copolymer was precipitated with excess
diethyl ether. The precipitate was dried under vacuum. The feed
Scheme 1 Schematic illustration of the formation of the PEG-SS-PLys-PLeu shell-
cross-linked (SCL) micelle and intracellular drug release.
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amounts of mPEG–NH
ESI, Table S1.†
2
, Lys(Z)-NCA and Leu-NCA are shown in held constant by adding 2 mL aqueous milieu aer each
sampling.
The amount of drug released from the micelles was
measured by UV at 369 nm. The amount of unloaded drug was
analyzed by measuring the absorbance of dialyzate aer drug
Deprotection of PEG-SS-PLys(Z)-PLeu
PEG-SS-PLys(Z)-PLeu (0.5 g) was dissolved in CF COOH (5 mL).
Subsequently a 33% solution of HBr in HAc (200 mL) was added,
3
loading. The cumulative drug release was calculated as follows:
and then the mixture was stirred under N
2
atmosphere for 2 h at
cumulative drug release ¼ (M /M ) ꢁ 100%
t
0
ꢀ
0
C. Aer that, the reaction mixture was precipitated with
excess diethyl ether and the precipitate was dried in vacuo.
where M
time t and M
PLeu micelles.
t
is the amount of drug released from the micelles at
0
is the amount of drug loaded in the PEG-SS-PLys-
Synthesis of FITC labeled PEG-SS-PLys-PLeu
The uorescein isothiocyanate (FITC) labeling of PEG-SS-PLys-
In vitro cytotoxicity study
32
PLeu was performed by a method published elsewhere. PEG-
SS-PLys-PLeu and FITC were dissolved in 5 mL anhydrous DMF
and then triethylamine was added; the reaction was allowed to
stand at room temperature for 48 h. The mixture was dialyzed
3
HeLa cells were cultured in 96-well plates (5 ꢁ 10 cells per well
with 100 mL of DMEM, supplemented with 10% fetal bovine
serum and 0.1% penicillin–streptomycin) for 24 h. Then the
cultured medium was replaced by DMEM containing PEG-SS-
PLys-PLeu copolymers with varying concentrations. Aer the
cells were incubated for 48 h, the medium with the polymers
was replaced by fresh DMEM, and 20 mL of MTT solution (5 mg
(
MWCO ¼ 3500 Da) against distilled water and the water was
refreshed three times everyday to remove the excess FITC. The
resulting product was freeze-dried.
ꢂ1
mL ) was added and the cells were incubated for 4 h. Then, the
Micelle formation and shell cross-linking
medium was removed and 150 mL of DMSO was added to
PEG-SS-PLys-PLeu (2 mg) was dissolved in DMF (4 mL) and put dissolve the formed crystals. The optical density (OD) was
into a dialysis tube (MWCO ¼ 8000–14 000 Da). DMF was measured at 492 nm with Multiskan MK3 (Thermo Fisher
removed by dialysis against deionized water, during which the Scientic, Waltham, MA, USA). The cell viability was calculated
water was refreshed every 12 h.
Shell cross-linking was carried out by adding 3,3 -dithiodi-
propionic acid as a linker. Firstly, 3,3 -dithiodipropionic acid
as follows:
0
0
cell viability (%) ¼ (ODtreated/ODcontrol) ꢁ 100%
was reacted with EDC and NHS for 8 h, and then the mixture
was added to the solution of PEG-SS-PLys-PLeu micelles at pH
where ODcontrol was obtained in the absence of polymer and
ODtreated was obtained in the presence of polymer.
5.0. The reaction mixture was stirred for 12 h at room temper-
ature. Aer dialysis against deionized water for 12 h to remove
the unreacted reagent, the dialyzate was lyophilized to obtain
the shell cross-linked polymer micelles.
Cell internalization of the micelles
HeLa cells were cultured in a 6-well plate at a concentration of
5
1
ꢁ 10 cells per well in DMEM supplemented with 10% fetal
ꢀ
bovine serum and 0.1% penicillin–streptomycin at 37 C in
Drug loading and in vitro drug release
2
humidied air/5% CO . Aer incubation for 24 h, the culture
medium was removed and replaced with DMEM containing
PEG-SS-PLys-PLeu micelles (500 mg L ), and incubated for 24
h. Aer incubation, the medium was removed and cells were
rinsed twice with DPBS, and then 4% paraformaldehyde was
added and incubated at room temperature for 15 min. Aer
being rinsed twice with DPBS, the cells were observed by
confocal laser scanning microscopy (Nikon C1-si, Japan).
The excitation wavelength was 488 nm, and the uorescence
emission was detected at 600–650 nm.
PEG-SS-PLys-PLeu (18 mg) and camptothecin (CPT) (6 mg) were
dissolved in DMF (36 mL). The unloaded CPT and the organic
ꢂ1
solvent were removed by dialysis against 2 L deionized water for
ꢀ
2
8
4 h at 25 C (MWCO ¼ 3500 Da). The water was refreshed every
h. Aer dialysis, the solution in the dialysis tube was collected
and divided into three parts.
One part was divided into three equal portions, and then the
three samples were respectively immersed in 150 mL of PBS
buffer without GSH, with 1 mM GSH and with 10 mM GSH.
Aliquots of 2 mL were withdrawn from the solution periodically.
The volume of the solution was held constant by adding 2 mL
aqueous milieu aer each sampling.
The other two parts were reacted with the linkers for 12 h, HeLa cells were seeded in 96-well plates (5 ꢁ 10 cells per well)
and then dialyzed against deionized water for 12 h to remove for 24 h. The cells were then treated with GSH-OEt for 4 h. Aer
the unreacted reagent. Then the solution was divided into three washing off the GSH-OEt, DMEM containing CPT-loaded
Cell growth inhibition measurements
3
equal portions and then the three samples were respectively micelles with varying concentrations was added. The cells were
ꢀ
immersed in 150 mL of PBS buffer without GSH, with 1 mM incubated at 37 C in 5% CO atmosphere for 48 h, and then the
2
GSH and with 10 mM GSH. Aliquots of 2 mL were withdrawn culture medium was replaced by fresh DMEM, and 20 mL of
ꢂ1
from the solution periodically. The volume of the solution was MTT solution (5 mg mL ) was added to the cells. Aer
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incubation for 4 h, 150 mL of DMSO was added to dissolve the
formed crystals. The cytotoxicity was evaluated as described
previously.
Results and discussion
Synthesis of PEG-SS-PLys-PLeu copolymer
In this work, the triblock copolymer PEG-SS-PLys-PLeu was
designed and synthesized. The synthetic route is shown in
Scheme 2. PEG-SS-NH was prepared by conjugating cystamine
2
to mPEG–COOH using DCC/NHS coupling, and then PEG-SS-
PLys(Z) and PEG-SS-PLys(Z)-PLeu were sequentially synthesized
using ring-opening polymerization of Lys(z)-NCA and Leu-NCA,
initiated by the primary amine end-groups on the polymer
chains. Due to poor solubility of the poly(L-leucine) segments in
33
common organic solvents, poly(rac-leucine) was chosen as a Fig. 2 FT-IR spectra of (a) PEG-SS-PLys(Z), (b) PEG-SS-PLys(Z)-PLeu, and (c) PEG-
substitute to improve the solubility of the copolymers. PEG-SS- SS-PLys-PLeu.
PLys-PLeu was obtained aer the subsequent deprotection
process in the presence of HBr in HAc. The copolymers with
different feed weight ratios were coded as P1 and P2 respec- complete removal of the benzyloxycarbonyl groups. The FT-IR
tively, as shown in ESI, Table S1.†
The structure of the polymers was characterized by H NMR I and II bands of PLys(Z) units at 1667 and 1549 cm , the
and FT-IR spectroscopy. As shown in Fig. 1, the peaks at 7.2 ppm stretching vibration absorbance of the C]O at 1708 cm and
spectra of the copolymers are shown in Fig. 2. The typical amide
1
ꢂ1
ꢂ1
ꢂ1
and 5.2 ppm were assigned to the protons on the benzene the stretching variation absorbance of N–H at 3304 cm are
ring and the methylene of the benzyloxycarbonyl groups. The shown in Fig. 2a. With the introduction of PLeu blocks, the
resonance peaks at 3.2 and 1.2 ppm from the methylene protons typical amide I and II bands were strengthened as compared
ꢂ1
ꢂ1
of PLys(Z) blocks and the peak at 0.9 ppm from the methyl with the bands at 1708 cm . In Fig. 2c, the band at 1708 cm
protons of PLeu blocks demonstrated the successful synthesis has completely disappeared, which indicates that the depro-
of PEG-SS-PLys(Z)-PLeu. The peaks at 7.2 and 5.2 ppm tection process was successful. The molar ratios of EG units to
completely disappeared aer deprotection, which indicated the Lys units and Leu units in the copolymer could be calculated
1
based on H NMR spectra, which were 113 : 7 : 20 for P1 and
1
13 : 7 : 11 for P2. The number-average molecular weight
1
determined by GPC was slightly lower than that calculated by H
NMR, probably attributable to the rod-like PLeu segments.
Micelle formation and characterization
In an aqueous solution, PEG-SS-PLys-PLeu copolymers can self-
assemble into micelles by a dialysis method. The obtained
micelles possessed three domains: the PEG blocks formed the
outer corona, the PLys segments formed the middle shell with
reactive primary amines, and the hydrophobic PLeu blocks
formed the inner core. The critical micelle concentration (CMC)
was estimated using pyrene as the uorescence probe, shown in
ESI, Fig. S1.† The CMC values of P1 and P2 were 49 and 150 mg
ꢂ1
L
, respectively. Since P1 micelles were more stable, further
studies were carried out based on P1.
The morphology and size distribution of micelles were
studied by TEM, SEM and DLS. The TEM image (Fig. 3a) showed
that the micelles had a regular spherical shape, and dispersed
very well. The mean diameter was around 150 nm, which was
consonant with the results determined by DLS. For shell-cross-
linked (SCL) micelles, the PLys middle shell was cross-linked
0
with a disulde-containing cross-linker (3,3 -dithiodipropionic
acid) using EDC/NHS coupling in aqueous solution at pH 5.
Fig. 3c shows the TEM image of SCL micelles. The SCL micelles
were well dispersed and slightly smaller compared with the
1
Fig. 1
H NMR spectra of (I) PEG-SS-PLys(Z), (II) PEG-SS-PLys(Z)-PLeu, and (III)
PEG-SS-PLys-PLeu.
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decrease in scattering intensity was much slower. The results
indicate that the shell cross-linking could greatly improve the
structural stability of the micelles.
Drug release property of PEG-SS-PLys-PLeu micelles
The inuence of PEG shedding and shell cross-linking on the
drug release behavior of the micelles was studied through the
comparison of the release proles of NCL micelles, cleavable
SCL micelles (SCM1) and uncleavable SCL micelles (SCM2). The
0
shells of SCM1 and SCM2 were cross-linked by adding 3,3 -
dithiodipropionic acid or hexanedioic acid, respectively.
Camptothecin (CPT) was used as a hydrophobic model drug.
CPT loading was performed by dialysis. The drug loading in
NCL micelles was 18 wt% with an encapsulation efficiency of
54%, while the drug loading in SCM1 (12 wt%) and SCM2 (14 wt
%
) was lower because of the loss during the succedent shell
Fig. 3 TEM image (a) and size distribution (b) of the non-cross-linked micelles in
an aqueous medium; TEM image (c) and size distribution (d) of the shell-cross-
linked micelles in an aqueous medium. The scale bar is 200 nm.
cross-linking process.
The release proles of the micelles are shown in Fig. 5. In the
absence of GSH, compared with NCL micelles, the drug release
rates from SCM1 and SCM2 were slower. Clearly, the cross-
non-cross-linked (NCL) micelles, which was consistent with the
hydrodynamic radius (Rh) of the SCL micelles. These results
indicated that the middle shells were compacted as a result of
chemical network formation. Further verication of the shell
cross-linking of the micelles was conrmed by the zeta potential
(z). The zeta potential (z) of the non-cross-linked (NCL) micelles
was positive (z ¼ 12.18 mV) due to the primary amines on the
PLys segments, while that of the SCL micelles was slightly
negative (z ¼ ꢂ3.52 mV), which may be attributed to excess
carboxyl groups from the cross-linker.
To investigate whether the shell cross-linking could enhance
the structural stability of the micelles, we observed the behavior
of the SCL micelles in severe environments. In THF, the NCL
34
micelles should collapse, whereas the SCL micelles should be
able to sustain the nanostructure. So we dissolved the lyophi-
lized SCL micelles in THF. As shown in the SEM image (Fig. 4a),
many assemblies could still be observed in the photograph,
which demonstrates that the shell cross-linking of the micelles
was successful. We also diluted aqueous solutions containing
SCL micelles and NCL micelles with DMF at different concen- Fig. 5 Drug release profiles of (a) NCL micelles, (b) cleavable SCL micelles (SCM1)
trations and determined their scattering intensity. As illustrated and (c) uncleavable SCL micelles (SCM2). The dependence of the scattering
intensity of the micelles containing solution on the percentage of DMF (d).
in Fig. 5d, with an increase in the DMF component, the NCL
micelle dissociated, leading to a sharp decrease in scattering
intensity, while the SCL micelle was more stable and the
Fig. 6 (a) Cytotoxicity of the polymers and the SCL micelles at different
concentrations for HeLa cells; (b) cytotoxicity of CPT-loaded SCL micelles for HeLa
Fig. 4 SEMimage(a)andsizedistribution(b)oftheshell-cross-linkedmicellesinTHF.
cells without pretreatment and with pretreatment with 10 mM GSH-OEt.
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linked shell retarded the drug diffusion. This result is in disulde exchange reaction occurred fast. If the concentration
35
accordance with the literature. In the presence of 1 mM GSH, of GSH was further increased to 10 mM, the release rates from
the drug release from all the micelles became faster, and the both NCL and SCM1 micelles would be drastically increased,
drug release from the NCL micelles was still slower than that and the encapsulated drug could be completely released out in
from both types of SCL micelles (SCM1 and SCM2). For the SCL several hours. Interestingly, the release behavior of SCM2 was
micelles, the disulde bonds were embedded within the quite different. With an increase in GSH concentration, in the
hydrophobic–hydrophilic interface of the micelles in aqueous initial release stage, the drug release rate decreased. A possible
solution, and the bond cleavage by the hydrophilic reducing reason was that the shell shedding of the micelles was so
agent GSH was not so fast. In the NCL micelles, the disulde abrupt, and the drug did not have sufficient time to be released
bonds were located in the hydrophilic corona–shell interface, out before the aggregation of SCM2 micelles.
and GSH could access the bonds more easily so the thiol–
Cell growth inhibition measurements
The cell cytotoxicity of the PEG-SS-PLys-PLeu copolymer to HeLa
cells was estimated using an MTT assay to evaluate the
biocompatibility of the copolymer. As shown in Fig. 6a, the PEG-
SS-PLys-PLeu copolymer exhibited a detectable cytotoxicity,
presumably due to the primary amine groups in the chains.
Aer shell cross-linking, most of the primary amine groups on
the middle PLys segments were converted to amide linkages, so
the SCL micelles did not show an apparent inhibition effect on
cell viability. The low cytotoxicity of SCL micelles is favorable for
their practical applications in drug delivery.
To assess the redox-responsive functions of the SCL micelles
in living cells, the cell growth inhibition effect of drug-loaded
micelles was further evaluated. With an increase in GSH
concentration, the drug release was accelerated, leading to an
enhancement of cytotoxicity. For the cells with pretreatment,
HeLa cells were incubated with glutathione monoester (GSH-
OEt) for 4 h. GSH-OEt, a glycine esteried form of GSH,
can easily penetrate the cellular membranes and is rapidly
Fig. 7 CLSM images of HeLa cells co-incubated with PEG-SS-PLys-PLeu. (a) FITC
hydrolyzed in the cytoplasm to generate high intracellular
GSH concentration. Then, HeLa cells were incubated with
fluorescence in cells (green); (b) cell nuclei stained by Hoechst 33258 (blue); (c)
overlays of images (a) and (b); (d) phase contrast.
Fig. 8 CLSM images of HeLa cells co-incubated with CPT-loaded SCL micelles. (a and b) Non-pretreated with GSH-OEt and co-incubated for 3 h, (a) CPT fluorescence in
cells (blue) and (b) phase contrast; (c and d) non-pretreated with GSH-OEt and co-incubated for 6 h, (c) CPT fluorescence in cells (blue) and (d) phase contrast; (e and f)
pretreated with GSH-OEt and co-incubated for 3 h, (e) CPT fluorescence in cells (blue) and (f) phase contrast; (g and h) pretreated with GSH-OEt and co-incubated for
6
h, (g) CPT fluorescence in cells (blue) and (h) phase contrast.
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Conclusions
In summary, we prepared a series of novel shell cross-linked
micelles with detachable PEG corona, which self-assembled
from PEG-SS-PLys-PLeu copolymers. The micelles were stable in
PBS buffer, while they underwent destruction of the cross-
linked shell with detachment of PEG corona in the presence of
GSH due to the cleavage of disulde bonds. The cell internali-
zation experiment conrmed that the micelles could be
successfully internalized into HeLa cells. The in vitro drug
release study showed that the micelles exhibited reduced drug
loss in extracellular environments and accelerated drug release
at the cytoplasmic GSH level. These glutathione-responsive
micelles have great potential in intracellular drug delivery.
Fig. 9 Flow cytometric analysis of non-pretreated and GSH-OEt pretreated HeLa
cells incubated with CPT-loaded micelles for 4 h.
Acknowledgements
This work was nancially supported by the Ministry of Science
CPT-loaded SCL micelles. The cell viability is shown in Fig. 6b. and Technology of China (2011CB606202), National Natural
Noticeably, the cell viability of the cells pretreated with GSH-OEt Science Funds for Distinguished Young Scholar (51125014),
was much lower than that of the cells without pretreatment, Program for Changjiang Scholars and Innovative Research
which illustrated that the CPT-loaded SCL micelles showed Team in University (IRT1030) and the Fundamental Research
remarkably enhanced drug release in the cells pretreated with Funds for the Central Universities.
GSH-OEt.
Notes and references
Confocal laser scanning microscopy observation
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