Dual-vectors of anti-cancer drugs and genes based on pH-sensitive micelles self-assembled from hybrid polypeptide copolymers

Article abstract of DOI:10.1039/c0jm03385e

A series of amphiphilic pH-sensitive hybrid polypeptide copolymers, poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-phenylalanine) (PEG-PLL-PLP) were synthesized. The copolymers could self-assemble into micelles with PLP as the hydrophobic core and PEG-PLL as the hydrophilic shell, as evidenced by ¹HNMR and TEM. These micelles exhibited obvious pH response in hydrodynamic diameter and pH-dependent drug release behavior, attributed to the protonation/deprotonation of amino groups in PLL segments. The copolymers could further condense plasmid DNA efficiently. Importantly, the polymer/DNA complexes showed high transfection efficiency in 293T cells under optimized conditions. This study suggested the copolymers may have great potential in both drug and gene delivery. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c0jm03385e

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links <
C
Journal of
Materials Chemistry
Cite this: J. Mater. Chem., 2011, 21, 3100
www.rsc.org/materials
PAPER
Dual-vectors of anti-cancer drugs and genes based on pH-sensitive micelles
self-assembled from hybrid polypeptide copolymers
ab
a
a
a
a
a
Yong-Yong Li,† Shou-Hu Hua,† Wang Xiao, Hui-Yuan Wang, Xiao-Hua Luo, Cao Li, Si-Xue Cheng,
a
a
a
Xian-Zheng Zhang* and Ren-Xi Zhuo
Received 8th October 2010, Accepted 30th November 2010
DOI: 10.1039/c0jm03385e
A series of amphiphilic pH-sensitive hybrid polypeptide copolymers, poly(ethylene glycol)-b-poly-
(
L-lysine)-b-poly(L-phenylalanine) (PEG-PLL-PLP) were synthesized. The copolymers could self-
assemble into micelles with PLP as the hydrophobic core and PEG-PLL as the hydrophilic shell, as
1
evidenced by HNMR and TEM. These micelles exhibited obvious pH response in hydrodynamic
diameter and pH-dependent drug release behavior, attributed to the protonation/deprotonation of
amino groups in PLL segments. The copolymers could further condense plasmid DNA efficiently.
Importantly, the polymer/DNA complexes showed high transfection efficiency in 293T cells under
optimized conditions. This study suggested the copolymers may have great potential in both drug and
gene delivery.
1
4
Introduction
synthesis of polypeptide hybrid block copolymers. These
copolymers have potential to be biocompatible and biodegrad-
able polymer-based carriers used in DDSs and GDSs because of
their chemical and structural similarity to natural biomolecules
such as proteins. Several kinds of polypeptides used in DDSs or
Over the past decade, abundant attention has been paid to
investigating the properties and applications of various self-
1
–4
assemblies in aqueous solution. Micelles, self-assembled from
amphiphilic block copolymers, are certainly one of the most
promising nanostructures, for their biomedical applications as
15
GDSs have been reported, like poly(L-lysine), poly(L-gluta-
9
16
17
mate),
Laemmli
poly(L-lysine) (PLL) to condense DNA in 1975, PLL was widely
poly(L-aspartate)
and poly(L-leucine).
Since
5,6
drug delivery systems (DDSs). Generally, hydrophobic anti-
cancer drugs could be loaded in the hydrophobic cores of the
micelles, while plasmid DNA (pDNA) could be condensed by the
cationic micelles in gene delivery systems (GDSs). However,
polymer-based carriers in DDSs and GDSs have been extensively
18
demonstrated the exceptional capability of
19–22
used as in vitro and in vivo GDSs,
due to the abundant amino
groups on the side chains of PLL. In GDSs, to achieve a high
efficiency of condensing DNA, the molecular weight of PLL is
usually above 3000 Da. However, high molecular weight PLL
7,8
studied separately. To our knowledge, there were limited
reports on the carriers that could be used in both DDSs and
23
exhibits relatively high toxicity, as well as the penalty of the
9–11
GDSs,
which may overcome the limitations of many mono-
high tendency of PLL/DNA complexes to aggregate or even
24
precipitate depending on the ionic strength of the solution. To
therapies by attacking the disease system on multiple fronts, due
to that multi-target therapeutics can be more efficient and less
vulnerable to adaptive resistance because the biological system is
less able to compensate for the action of two drugs simulta-
overcome these defects of high molecular weight PLL,
poly(ethylene glycol) (PEG), a widely used polymer with good
biocompatibility, non-toxicity as well as ease of excretion from
living organisms, is commonly used to modify the PLL based
vectors. For example, Kim and coworkers synthesized biode-
gradable multiblock copolymers for gene delivery based on PEG
12
neously.
As for DDSs and GDSs, the biocompatibility and biode-
gradability of the polymer-based carriers are always crucial
issues, with regard to the risks brought by the accumulation of
non-degradable polymers in the body and the interaction with
25
and PLL. Their results showed improved transfection efficiency
without adversely affecting the cell viability.
13
the host cells and tissues, which may cause a long-term toxicity.
In our research, we designed and synthesized a new type of
hybrid polypeptide micelle based on poly(ethylene glycol)-b-
poly(L-lysine)-b-poly(L-phenylalanine) (PEG-PLL-PLP) for
both drug and gene delivery applications. All of the polymer
blocks in this system have good biocompatibility. The in vitro
drug release of drug loaded PEG-PLL-PLP micelles was studied
under different pHs, and the ability of the copolymer to condense
pDNA and to mediate gene expression was evaluated. The
In the mid 1970s, Gallot and coworkers first reported the
a
Key Laboratory of Biomedical Polymers of Ministry of Education &
Department of Chemistry, Wuhan University, Wuhan, 430072, P. R.
China. E-mail: xz-zhang@whu.edu.cn
b
The Institute for Advanced Materials and Nano Biomedicine (iNANO),
Tongji University, Shanghai, 200092, P. R. China
†
Authors contributed equally to this work.
3
100 | J. Mater. Chem., 2011, 21, 3100–3106
This journal is ª The Royal Society of Chemistry 2011

View Article Online
results showed these cationic micelles were able to deliver both
drug and gene.
LP–NCA initiated by PEG–PLL(Z) in DMF with a feed ratio of
2.5 : 1, 1 : 1, 1 : 2 (weight ratio of PEG–PLL(Z) to LP–NCA),
respectively. The resultant solution of LP–NCA and PEG–
ꢀ
PLL(Z) was stirred at 30 C for 72 h under N
2
atmosphere and
Experimental
then the reaction mixture was poured into a large excess of
diethyl ether to precipitate the PEG–PLL(Z)-PLP copolymer.
The obtained product was purified by repeated precipitation in
diethyl ether and dried in a vacuum. The purified yields of P1, P2
and P3 were 86.7%, 75.1% and 53.8%, respectively.
Materials
0
O-(2-aminoethyl)-O -methylpolyethylene glycol (PEG-NH
2
, Mw ¼
2
000) was purchased from Sigma Chemical Co. and used without
further purification. 3-Benzyloxycarbonyl-L-lysine (LL(Z)) and
L-phenylalanine (LP) were purchased from GL Biochem
Deprotection of PEG–PLL(Z)–PLP triblock copolymers
(
Shanghai) Ltd. Hydrogen bromide 33 wt% solution in glacial
acetic acid was purchased from ACROS Organics. Tetrahydro-
furan (THF) and n-hexane were of analytical grade and
dried with sodium to remove water before use.
PEG–PLL–PLP (C) was obtained by the deprotection of PEG–
PLL(Z)–PLP. 320 mg PEG–PLL(Z)–PLP was dissolved in
1
0 mL of trifluoroacetic acid, then 4 equiv of a solution of 33 wt%
0
N,N -Dimethyformamide (DMF) was obtained from Shanghai
HBr in HAc with respect to the benzyl carbamate (Z) groups
ꢀ
Chemical Reagent Co., China and used after distillation under
reduced pressure. Triphosgene was recrystallized from diethyl
ether before use. All other solvents were of analytical grade and
used without further purification.
were added and the solution was stirred at 0 C for 1 h under
nitrogen. After precipitation with an excess of diethyl ether, the
crude product was dissolved in DMF and the solution was dia-
lyzed against pH 9.0 ammonia solution for 48 h using a dialysis
membrane (MWCO ¼ 3500 Da) to remove the HBr attaching on
PLL segment. Another exhaustive dialysis against deionized
water was conducted for 24 h before the resulting product was
collected by freeze-drying. PLL homopolymer was synthesized
by using dodecylamine as the initiator under the similar synthesis
condition.
Synthesis of 3-benzyloxycarbonyl-L-lysine N-carboxyanhydride
(
(
LL(Z)–NCA) and L-phenylalanine N–carboxyanhydride
LP-NCA)
The following synthesis procedure was adapted from the litera-
26
ture. Typically to a dry THF suspension (90 mL) of LL(Z)
7.6 g, 27 mmol) or LP (4.5 g, 27 mmol), a THF solution (10 mL)
(
FITC Functionalization of PEG–PLL–PLP
of triphosgene (3.4 g, 11 mmol) was dropwise added and the
ꢀ
mixture was stirred at 50 C under N atmosphere. The reaction
2
PEG–PLL–PLP was fluorescently labeled with fluorescein iso-
thiocyanate (FITC). P1 (20 mg) and FITC (7 mg) were dissolved
in 5 mL of DMF. The reaction was performed for 48 h at room
was stopped when the suspension turned to a clear solution. The
solution was poured into excess dried n-hexane to obtain crude
crystals of LL(Z)–NCA or LP-NCA, which were further
recrystallized from dried THF/n-hexane twice and dried in
vacuum. The purified yields of LL(Z)–NCA and LP–NCA were
ꢀ
temperature (18 C). For purification, the resultant solution was
dialyzed (molecular weight cut-off (MWCO) ¼ 3500 Da) against
water (the water was refreshed periodically to remove the excess
FITC). Finally, the resulting product was collected by freeze-
drying.
7
.9 g (Yield: 95.7%) and 4.7 g (Yield: 91.5%), respectively.
LL(Z)–NCA (NMR): 7–7.5 ppm (m, 5H, C ), 5.0–5.2 ppm (s,
O), 4.2–4.5 ppm (t, 2H, OCCH NH–), 3.1–
.3 ppm (t, 2H, –NHCH CH –), 1.0–2.0 ppm (m, 6H,
CH CH CH –); LP–NCA (NMR): 7–7.5 ppm (m, 5H, C H ),
6 5
H
2
3
–
4
–
H, C
6
H
5
CH
2
2
2
2
Measurements
2
2
2
6
5
1
.7–5.0 ppm (t, 2H, OCCH NH–), 2.8–3.2 ppm (m, 2H,
2
HNMR spectra were recorded on a Mercury VX-300 spec-
C H CH CH –).
6
trometer at 300M Hz using CF COOD and D O as the solvents.
3 2
5
2
2
Fluorescence spectra were recorded on a LS55 luminescence
spectrometer (Perkin-Elmer). The morphology of the micelles
was observed by a JEM-100CXa TEM at an acceleration voltage
of 100 kV. Nano-ZS ZEN3600 was used to determine the size
distribution of the self-assembled micelles and the z potential of
polymer/DNA complexes. The cellular internalization of the
micelles was studied by a confocal laser scanning microscope
(CLSM, Nikon C1-si, BD Laser) at 488 nm.
Synthesis of PEG–PLL(Z) copolymers
PEG–PLL(Z) (A) copolymers were prepared by ring opening
polymerization of LL(Z)–NCA initiated by PEG–NH2 in
DMF with a feed ratio of 1 : 1.5 (weight ratio of PEG–NH to
2
LL(Z)–NCA). The resultant solution of LL(Z)–NCA and
ꢀ
PEG–NH was stirred at 30 C for 72 h under N atmosphere
2
2
and then the reaction mixture was poured into a large excess of
diethyl ether to precipitate the PEG–PLL(Z) copolymer. The
obtained product was purified by repeated precipitation in
diethyl ether and dried in a vacuum. The purified yield was
In vitro cytotoxicity study
1
00 mL of cancer cells (HeLa) in DMEM were added to each well
7
ꢂ1
of a 96-well plate (concentration ¼ 6 ꢁ 10 cells L ). The
9
1.6%.
3
number of cells in each well was z 6 ꢁ 10 . After incubation for
ꢀ
2
4 h in an incubator (37.0 C, 5% CO ), the culture medium was
2
Synthesis of PEG–PLL(Z)–PLP triblock copolymers
transferred to 100 mL of DMEM containing the PEG–PLL–PLP
with varying concentrations, and the mixture was further incu-
bated for 24 h. Then, DMEM with the polymer was replaced by
Similarly, PEG–PLL(Z)–PLP (B) triblock copolymers (P1, P2,
P3) were synthesized by ring opening polymerization of
This journal is ª The Royal Society of Chemistry 2011
J. Mater. Chem., 2011, 21, 3100–3106 | 3101

View Article Online
ꢂ1
fresh DMEM and 20 mL of a MTT solution (5 mg mL ) was
loading was measured by UV spectroscopy to determine the
amount of unloaded drugs. It was found that around 14.8 wt% of
DOX was loaded into PEG-PLL-PLP micelles.
added to the cells. After incubation for 4 h, 200 mL of DMSO was
added and the mixture was shaken at room temperature. The
optical density (OD) was measured at 570 nm with a Microplate
Reader Model 550 (BIO-RAD, USA). The viable rate was
calculated by the following equation: Cell viability (%) ¼
After dialysis, the dialysis tube was directly immersed into
400 mL of buffer solution with different pHs. Aliquots of 2 mL
were withdrawn from the solution periodically. The volume of
solution was held constant by adding 2 mL aqueous milieu after
each sampling. The amount of DOX released from micelles was
measured by UV absorbance. The cumulative drug release was
calculated from the following relationship: Cumulative drug
release (%) ¼ (M /M ) ꢁ100, where M is the amount of drug
(
ODtreated/ODcontrol) ꢁ100, where ODcontrol was obtained in the
absence of polymer and ODtreated was obtained in the presence of
polymer.
Micelle formation
t
0
t
released from micelles at time t, and M is the amount of
0
Micelles of PEG–PLL–PLP copolymer in aqueous media were
drug loaded into the PEG–PLL–PLP micelles. Here, M was
0
prepared by the membrane-dialysis method at room temperature
ꢀ
estimated by subtracting the amount of unloaded drug from the
feed drug amount (1 mg).
(
18 C). PEG–PLL–PLP (4 mg) was dissolved in a mixture of
DMF (4 mL) with rapid stirring. After that, the solution was
subjected to dialysis against deionized water for 24 h using
a dialysis membrane (MWCO ¼ 3500 Da). The water was
refreshed every 12 h to remove all DMF from the sample.
In vitro transfection
The PEG–PLL–PLP micelles were dissolved into PBS solution at
various concentrations and then added to PGL-3 plasmid DNA
to obtain polymer/DNA complexes with different w/w ratios.
Fluorescence measurements and determination of CMC
ꢀ
The complexes were vortexed and incubated for 30 min at 37 C
Pyrene was used as a hydrophobic fluorescent probe. Aliquots of
ꢂ6
before the in vitro transfection experiment. Transfection studies
pyrene solutions (6 ꢁ 10 M in acetone, 1 mL) were added to
were performed with 293T cells, which were seeded at a density of
4
containers, and acetone was allowed to evaporate. Ten-millilitre
aqueous polymer solutions at different concentrations were then
added to the containers containing the pyrene residue. It should
6
ꢁ 10 cells/well in a 24-well plate and cultured 48 h in 1 mL of
DMEM. PGL-3 plasmid DNA was used as the reporter gene to
evaluate the transfection efficiency. The PEG–PLL–PLP/PGL-3
complexes were prepared at N/P ratios ranging from 20 to 130.
The PEG–PLL–PLP/PGL-3 complexes were prepared at each
optimal w/w ratio and PEI (25 kDa, branched) was used for
be noted that the concentration of the pyrene in every tube was
ꢂ7
6
ꢁ 10 M, which would be adequate for the critical micelle
concentration (CMC) measurements. The solutions were kept at
room temperature for 24 h to reach the equilibrated solubiliza-
tion of pyrene in the aqueous phase. Emission spectra were
recorded ranging from 350 to 600 nm with an excitation of
comparison. The complexes in serum-free DMEM were added to
ꢀ
2
93T cells for 4 h at 37 C, then the serum-free DMEM was
replaced by fresh DMEM and the cells were further incubated for
8 h.
A chemiluminometer (Lumat LB9507, EG&G Berthold,
3
37 nm. Both excitation and emission bandwidth were 5 nm.
4
From the pyrene emission spectra, the intensities at 393 nm were
analyzed as a function of the polymer concentrations. A CMC
value was determined from the intersection of the tangent to the
curve at the inflection with the horizontal tangent through the
points at low concentration.
Germany) was used to measure relative light units (RLUs). The
total protein content measured by the BCA protein assay kit was
analyzed to normalize the RLUs and the results were expressed
ꢂ1
as RLU mg protein.
Agarose gel retardation assay
Cell internalization of PEG–PLL–PLP micelles
The agarose gel retardation assay was performed as follows. The
polymers were dissolved into PBS solution at various concen-
The behavior of the micelles entering the tumor cells was studied
by a confocal laser scanning microscopy (CLSM) at 488 nm. A
trations and then added to 1 mL of plasmid EGFP-C1 DNA
ꢂ1
ꢀ
typical process was as follows: HeLa cells, maintained at 37 C,
(
100 ng mL in 40 mM Tris-HCl buffer solution) to make
5
% CO2 in DMEM medium, were chosen to assess the cell
mixtures of 5 mL. The complexes were vortexed and incubated
ꢀ
internalization into the tumor cells. After incubation for 24 h in
an incubator, the culture medium was replaced by medium
for 30 min at 37 C. Then the complexes were loaded onto the
0
.7% (W/V) agarose gel containing GelRedꢀ. Electrophoresis
was carried out with a current of 80 V for 80 min in tris-acetate
TAE) running buffer. DNA was visualized with a UV lamp
ꢂ1
containing P1 (1 mg mL ) and the mixture was further incu-
bated for 4 h. Cellular internalization was studied after the plates
being washed with PBS for six times.
(
using a GelDoc system (Imago).
Results and discussion
Drug loading and in vitro drug release
Synthesis of PEG–PLL–PLP
PEG–PLL–PLP (2 mg) and doxorubicin (DOX, 1 mg) were
dissolved in 2 mL of DMF. The solution was placed into a dial-
Hybrid polypeptide block copolymers were synthesized by ring
opening polymerization initiated by amino-terminated synthetic
polymers, in which the initiator amine underwent a nucleophilic
addition to the C-5 carboxy group of the amino acid-NCA,
ysis tube (MWCO ¼ 3500 Da) and subjected to dialysis against
ꢀ
1
000 mL of distilled water for 24 h at 18 C. The water was
refreshed after 12 h to remove the DMF. The dialyzate after drug
3
102 | J. Mater. Chem., 2011, 21, 3100–3106
This journal is ª The Royal Society of Chemistry 2011

View Article Online
called the primary amine mechanism, as proposed by
27
Goodman. In this study, PEG–NH was used as the initiator to
2
synthesize the PEG–PLL–PLP hybrid polypeptide copolymer.
The synthesis of PEG–PLL–PLP involved three steps as shown
in Fig. 1. First, LL(Z)–NCA and LP–NCA were prepared by
intramolecular ring closure of LL(Z) and LP, respectively. The
triblock copolymer B, protected by Z groups, was then synthe-
sized using successive ring opening polymerization. Finally, C
was obtained after the deprotection of B by HBr/HAc.
1
As shown in Fig. 2, HNMR proves the successful synthesis
and deprotection of PEG–PLL–PLP. Compared with Fig. 2A,
a new peak g appears at d ¼ 2.83 ppm in Fig. 2B, which is
assigned to the protons of the –CH – group in the PLP unit,
2
1
Fig. 2 HNMR spectra (II) of PEG–PLL(Z) (A), PEG–PLL(Z)–PLP
B) and PEG–PLL–PLP (C) in CF COOD.
indicating the successful synthesis of the triblock copolymer.
This is further confirmed by the increasing peak area ratio of
peak f (d ¼ 7.19 ppm, assigned to the protons of benzene group)
(
3
to peak a (d ¼ 3.81 ppm, assigned to the protons of the –CH
2
–
Micelle formation and characterization
group in PEG units). In Fig. 2C, peak e at d ¼ 5.0 ppm, attrib-
uted to the –CH – in Z group, totally disappears, suggesting
PEG–PLL–PLP micelles were obtained by a dialysis method.
The CMC of the copolymer was analyzed by fluorescent spec-
troscope using pyrene as a probe. From the plot of fluorescence
intensity versus copolymer concentration (Fig. 4I and Fig. 4II),
2
a thorough removal of Z groups. Other peaks are also illustrated
in Fig. 2. The products were designated P1, P2 and P3 respec-
tively in the order of the increasing feed of LP as shown in Table
ꢂ1
the CMC of the P1 and P2 obtained in this study are 375 mg L
ꢂ1
1
. Since the solubility of P3 in water or organic solvents is poor
and 139 mg L , respectively. Obviously, the values of CMC
due to the high content of LP units, we focused our study on P1
and P2.
decrease with the increasing hydrophobic block PLP content in
9
the copolymers, which is in agreement with earlier research.
TEM was engaged to investigate the morphologies of PEG–
PLL–PLP self-assemblies. As presented from the insert of
Fig. 4(III), well-dispersed PEG–PLL–PLP micelles with a regular
spherical shape and a diameter around 25 nm are formed, while
from the size distribution of PEG–PLL–PLP micelles
(Fig. 4(III)), the mean size of the micelles is around 45 nm. The
larger diameter from particle-size analyzer may be due to the
Cytotoxicity study
Cytotoxicity studies have been carried out in order to investigate
the biocompatibility of the PEG–PLL–PLP copolymer. The
results show that the PEG–PLL–PLP copolymers do not exhibit
apparent cytotoxicity when the copolymer concentration is
ꢂ1
below 500 mg L (Fig. 3), where the cell viability in the whole
28
window is all above 80% although with a little decrease as the
concentration increases, whereas PLL homopolymer with the
similar length of the PLL block in the copolymer (average 118
micelles with the internal structure of aggregates and the
micelles’ dehydration induced by water evaporation under
29
the high vacuum condition during TEM measurement.
1
units per PLL chain characterized by H NMR) displays obvious
The micellization behavior of PEG–PLL–PLP in aqueous
1
media was further confirmed by HNMR. The micelle suspension
lower cell viability at higher concentrations, indicating the
reduced cytotoxicitity of the block copolymer. Also from the
cytotoxicity data of the copolymers (P1 and P2) whose structures
differ only in the length of PLP, the copolymer with the longer
length shows a slightly higher cytotoxicity, indicating that PLP is
also an important factor in optimizing the properties of the
copolymer.
was freeze-dried to obtain PEG–PLL–PLP micelles. The
1
HNMR spectrum of the micelles in D O (Fig. 4(IV)) reveals that
2
the signal of protons in benzene groups of PLP units almost
disappears, suggesting a core-shell micellar structure of PEG–
PLL–PLP in D
O with isolated hydrophobic PLP inner cores
2
and hydrophilic PEG–PLL outer shells.
Fig. 1 Synthesis of the triblock polypeptide hybrid copolymer.
This journal is ª The Royal Society of Chemistry 2011 J. Mater. Chem., 2011, 21, 3100–3106 | 3103

View Article Online
Table 1 Characteristics of polymer molecular weight and composition
1
calculated from H NMR spectra
under different pHs were prepared. As shown in Fig. 5, by
increasing the pH from 4 to 10, the hydrodynamic diameter of
PEG–PLL–PLP micelles decreases apparently, i.e. around 15 nm
at pH 10 and around 60 nm at pH 4. This pH-induced size
variation is related to the ionization of amino groups in the PLL
segment. As we know, during the micellization of PEG–PLL–
PLP, the hydrophobic interactions between PLP blocks induce
Content of monomeric units in
mol%
Polymer
EG
LL
LP
M
n
P1
P2
45
45
96
96
16
37
15 000
18 000
Fig. 3 Cell viability of HeLa cells after 48 h incubation with PEG–PLL–
PLP (P1, P2) or PLL homopolymer.
pH response of the PEG–PLL–PLP micelles
Due to the amino groups on the side chains of the PLL segment,
PEG–PLL–PLP copolymers are expected to possess pH-sensitive
properties. To estimate the effect of environmental pH on the
hydrodynamic diameter of PEG-PLL–PLP micelles, micelles
Fig. 5 (a) Size distribution of PEG–PLL–PLP micelles at different pHs.
(b) Schematic illustration of the self-assembly of PEG–PLL–PLP
copolymers at different pHs.
Fig. 4 Intensity of the emission spectra at 393 nm as a function of the logarithm of the concentration of PEG–PLL–PLP (P1) (I) and PEG–PLL–PLP
ꢂ1
(P2) (II); size distribution (III) and TEM image (the insert) and of PEG–PLL–PLP micelles, the concentration of the copolymer was kept at 500 mg L
;
1
and HNMR spectrum of PEG–PLL–PLP micelles in D
2
O (IV).
3
104 | J. Mater. Chem., 2011, 21, 3100–3106
This journal is ª The Royal Society of Chemistry 2011

View Article Online
the microphase separation of intra-polymer chain with PEG–
PLL as the hydrophilic outer shell and PLP as the hydrophobic
inner core, respectively. When the micelles are prepared at lower
pH, more amino groups in PLL units could be protonated, which
means more positive charges appear in the hydrophilic shell of
the micelle, leading to an increased repulsion between PLL
segments and a larger hydrodynamic diameter.
Agarose gel retardation assay
Fig. 7 Particle size (a) and z potential (b) of PEG–PLL–PLP/DNA
The agarose gel retardation assay was performed to evaluate the
ability of PEG–PLL–PLP polymers to condense DNA. As
shown in Fig. 6, when PEG–PLL–PLP is bonded to DNA
completely, the weight ratios of P1 and P2 to DNA are 2 and 15,
respectively. Obviously, it is easier for P1 to condense DNA as
compared with P2, due to the higher density of amino groups of
P1. At the same weight ratios, there are more amino groups in P1
complexes in distilled water at various w/w ratios.
at the initial 20 h and then comes to a slow level. In comparison,
at pH 7 and pH 10 it shows nearly zero-order release. The
differences in drug release behaviors at different pHs are mainly
attributed to the change of the ionization extent of amino groups
in the PLL segment as discussed above. Apparently, repulsion
between ionized PLL chains at lower pH leads to larger size or
looser micelles, as also indicated by pH dependent size alter-
ations, making the drug diffuse out of the polymer matrix more
easily. Whereas at pH 7 and pH 10 PLL segment is in low ioni-
zation and tends to be more compact which is not favorable for
drug diffusion, it thus shows a moderate drug release velocity. As
a result, DOX-loaded PEG–PLL–PLP micelles exhibit a pH-
sensitive drug release behavior with a higher release rate at
a lower pH.
r
than P2, because of the higher M of P2 and the same number of
amino groups on each P1 and P2 chain. Hence, P1 demonstrates
a stronger ability to condense DNA.
Particle size and z potential measurements
The ability of PEG–PLL–PLP to condense DNA was also
evaluated by particle size and z potential measurements. As
shown in Fig. 7a, P1/DNA and P2/DNA complexes demonstrate
a similar trend in the variation of the particle size. With the
increasing of w/w ratios, the particle sizes of P1/DNA and P2/
DNA decrease, suggesting that DNA is not condensed
compactly at lower w/w ratios. The z potential of polymer/DNA
is presented in Fig. 7b. The z potential of P1/DNA keeps
constant at 27 mV at w/w ratios higher than 20, while the z
potential of P2/DNA rapidly increases with the enhancement of
w/w ratio from 10 to 30. The particle size and z potential
measurements indicate that the polymers could bind DNA to
form nanoparticles with positive surface charges, suggesting that
PEG–PLL–PLP/DNA complexes are suitable candidates for
gene transfection.
In vitro transfection
Gene transfection mediated by PEG–PLL–PLP/DNA complexes
was assessed in 293T cells in the absence of serum. Optimized w/
w ratio of 25 kDa PEI/DNA complex was used as the positive
control. As presented in Fig. 9, the P1/DNA complexes at N/P
ratio of 40 shows the highest transfection of all the ratios, though
lower to that of 25 kDa PEI/DNA complex at N/P ratio of 10 (or
w/w ¼ 1.3). In addition, the transfection efficiency of P1/DNA is
obviously higher than that of P2/DNA at the same N/P ratio,
which is probably attributed to the increased units of LP in P2
leading to a lower concentration of amino groups in micelles and
thus affecting the ability to condense DNA. As discussed above,
the amino density of P1 is higher than that of P2 when at the
same weight ratio, resulting in stronger ability to condense DNA
and higher transfection efficiency.
In vitro drug release
As is well known, hydrophobic drugs could be loaded in the
30
hydrophobic cores of the micelles. In this study, DOX, one of
the most common chemotherapeutic drugs, was chosen as the
hydrophobic model drug for its poor solubility and easy detec-
tion by fluorescence monitors. The applicability of PEG–PLL–
PLP micelles in controlled drug release was examined in buffer
solutions at different pHs. As shown in Fig. 8, the drug release at
pH 4 exhibits two-phase drug release, where burst release occurs
Fig. 8 Release behavior of DOX from DOX-loaded micelles at different
ꢀ
Fig. 6 Gel retardation assay of polymer/DNA complexes.
This journal is ª The Royal Society of Chemistry 2011
pHs at 37 C.
J. Mater. Chem., 2011, 21, 3100–3106 | 3105

View Article Online
Acknowledgements
We acknowledge the financial support from the Ministry of
Science and Technology of China (2009CB930300), Trans(New)-
Century Training Programme Foundation for the Talents from
the Ministry of Education of China, Shanghai Natural Science
Foundation (10ZR1432100) and Natural Science Foundation of
Hubei Province, China (2009CDA024).
References
Fig. 9 Luciferase expression in 293T cells transfected by PEG–PLL–
1 G. Riess, Prog. Polym. Sci., 2003, 28, 1107.
2 Z. Zhang, Z. Wei and M. Wan, Macromolecules, 2002, 35, 5937.
PLP/DNA complexes at N/P ratios ranging from 20 to 130.
3
4
5
6
7
8
9
H. Kukula, H. Schlaad, M. Antonietti and S. Forster, J. Am. Chem.
Soc., 2002, 124, 1658.
H. Wei, C. Y. Yu, C. Chang, C. Y. Quan, S. B. Mo, S. X. Cheng,
X. Z. Zhang and R. X. Zhuo, Chem. Commun., 2008, 38, 4598.
K. Kataoka, A. Harada and Y. Nagasaki, Adv. Drug Delivery Rev.,
2
001, 47, 113.
A. Lavasanifar, J. Samuel and G. S. Kwon, Adv. Drug Delivery Rev.,
002, 169.
2
J. Li, X. Ni, X. Li, N. K. Tan, G. T. Lim, S. Ramakrishna and
K. W. Leong, Langmuir, 2005, 8681.
T. G. Park, J. H. Jeong and S. W. Kim, Adv. Drug Delivery Rev., 2006,
58, 467.
H. Y. Tian, C. Deng, H. Lin, J. Sun, M. Deng, X. Chen and X. Jing,
Biomaterials, 2005, 26, 4209.
0 Y. Wang, S. Gao, W. H. Ye, H. S. Yoon and Y. Y. Yang, Nat. Mater.,
006, 5, 791.
1 J. L. Zhu, H. Cheng, Y. Jin, S. X. Cheng, X. Z. Zhang and
R. X. Zhuo, J. Mater. Chem., 2008, 18, 4433.
2 G. R. Zimmermann, J. L. Lehar and C. T. Keith, Drug Discovery
Today, 2007, 12, 34.
3 D. Fischer, T. Bieber, Y. Li, H. P. Elsasser and T. Kissel, Pharm. Res.,
1
1
1
1
2
Fig. 10 Confocal laser scanning images of HeLa cells treated with P1
micelles under excitation at 488 nm (a) and under bright field (b). The
ꢂ1
concentration of the copolymer was 500 mg L
.
1
999, 16, 1273.
14 B. Perly, A. Douy and B. Gallot, CR Acad Sci. Paris, 1974, 279C,
109.
15 P. Midoux and M. Monsigny, Bioconjugate Chem., 1999, 10, 406.
Cell internalization of PEG–PLL–PLP micelles
1
To examine whether the micelles could enter the tumor cells
1
6 K. Kataoka, T. Matsumoto, M. Yokoyama, T. Okano, Y. Sakurai,
S. Fukushima, K. Okamoto and G. S. Kwon, J. Controlled Release,
(
HeLa) for potential antitumor therapy, the cellular internali-
ꢂ1
zation of FITC functionalized micelles (500 mg L ) was inves-
tigated. After 4 h of incubation with the micelles, the HeLa cells
were observed by a CLSM and the result is demonstrated in
Fig. 10. It is found that the micelles could be internalized into the
HeLa cells because the green fluorescent intensity increases
suddenly inside HeLa cells.
2
7 S. H. Hua, Y. Y. Li, Y. Liu, W. Xiao, C. Li, F. W. Huang,
000, 64, 143.
1
X. Z. Zhang and R. X. Zhuo, Macromol. Rapid Commun., 2010, 31,
8
1.
1
1
8 U. K. Laemmli, Proc. Natl. Acad. Sci. U. S. A., 1975, 72, 4288–4292.
9 M. Ohsaki, T. Okuda, A. Wada, T. Hirayama, T. Niidome and
H. Aoyagi, Bioconjugate Chem., 2002, 13, 510.
2
2
2
0 J. M. Benns, J. S. Choi, R. I. Mahato, J. S. Park and S. W. Kim,
Bioconjugate Chem., 2000, 11, 637.
1 C. H. Ahn, S. Y. Chae, Y. H. Bae and S. W. Kim, J. Controlled
Release, 2004, 97, 567.
2 V. Stanic, Y. Arntz, D. Richard, C. Affolter, I. Nguyen, C. Crucifix,
P. Schultz, C. Baehr, B. Frisch and J. Ogier, Biomacromolecules, 2008,
Conclusions
In summary, a new type of hybrid polypeptide copolymer (PEG–
PLL–PLP) was synthesized. PEG–PLL–PLP copolymers could
self-assemble in water to form spherical micelles with a size
around 25 nm at neutral condition. A cell internalization
experiment exhibited that fluorescent micelles could be inter-
nalized into the cells. The micelles showed excellent controlled
drug release properties and apparent disparities in drug release
rates at different pHs. Cytotoxicity studies showed that the
copolymer exhibits good biocompatibility. In vitro transfection
showed that PEG–PLL–PLP/DNA complexes demonstrate
comparable transfection efficiency in 293T cells with lower
cytotoxicity as compared with PEI (25 KDa). This study suggests
that PEG–PLL–PLP could be used in both DDSs and GDSs.
9, 2048.
2
2
2
2
3 Y. H. Choi, F. Liu, J. S. Kim, Y. K. Choi, J. S. Park and S. W. Kim, J.
Controlled Release, 1998, 54, 39.
4 G. Liu, M. Molas, G. A. Grossmann, M. Pasumarthy, J. C. Perales,
M. J. Cooper and R. W. Hanson, J. Biol. Chem., 2001, 276, 34379.
5 M. Bikram, C. H. Ahn, S. Y. Chae, M. Lee, J. W. Yockman and
S. W. Kim, Macromolecules, 2004, 37, 1903.
6 W. H. Daly and D. Poche, Tetrahedron Lett., 1988, 29, 5859.
27 M. Goodman and J. Hutchison, J. Am. Chem. Soc., 1966, 88, 3627.
28 L. Zhang and A. Eisenberg, J. Am. Chem. Soc., 1996, 118, 3168.
2
9 Y. Hu, L. Zhang, Y. Cao, H. Ge, X. Jiang and C. Yang,
Biomacromolecules, 2004, 5, 1756.
3
0 K. Kataoka, G. S. Kwon, M. Yokoyama, T. Okano and Y. Sakurai,
J. Controlled Release, 1993, 24, 119.
3
106 | J. Mater. Chem., 2011, 21, 3100–3106
This journal is ª The Royal Society of Chemistry 2011