Fluorinated PEG-Polypeptide Polyplex Micelles Have Good Serum-Resistance and Low Cytotoxicity for Gene Delivery

Article abstract of DOI:10.1002/mabi.201700114

A novel PEGylation polypeptide, poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-cysteine) (PEG-PLL-PCys) triblock copolymer is synthesized via the sequential ring-opening polymerization of amino acid N-carboxyanhydrides initiated by methoxypolyethylene gl

Full text of DOI:10.1002/mabi.201700114

Macromolecular
Bioscience
Communication
Fluorinated PEG-Polypeptide Polyplex Micelles
Have Good Serum-Resistance and Low
Cytotoxicity for Gene Delivery
Ting Wu, Longhai Wang, Shenggang Ding,* Yezi You*
A novel PEGylation polypeptide, poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-cysteine) (PEG-
PLL-PCys) triblock copolymer is synthesized via the sequential ring-opening polymerization
of amino acid N-carboxyanhydrides initiated by methoxypolyethylene glycol amine (mPEG-
NH2, Mw is 2 kDa). Subsequently, the obtained polypeptide is partially conjugated with
fluorocarbon chains via disulfide exchange reaction. PLL seg-
ment can condense plasmid DNA through an electrostatic
force to form a complex core, PEG segment surrounding the
complex like a corona can prevent the complex from precipi-
tation and reduce the adsorption of serum, while PCys seg-
ment with fluorocarbon can enhance the cellular uptake and
the stability of the formed polyplex micelles in physiological
conditions. Experiment results exhibit that the fluorinated
polypeptides have low cytotoxicity and good gene trans-
fection efficiency even in the presence of 50% fetal bovine
serum.
1. Introduction
however, their further applications are limited by immu-
nogenicity,[5] toxicity, and difficulty of large-scale produc-
tion.[6] Recently, the principal focus of research is turned
to design effective nonviral vectors that condense and
protect deoxyribonucleic acid (DNA) for gene therapy. Cati-
onic lipids and polymers are promising gene carriers due
to their limited immunogenicity for clinical application
as well as the flexibility and diversity of their chemical
design.[7]
Gene therapy has attracted extensive attention during
the past three decades as a promising approach for the
treatment of a broad variety of human diseases, ranging
from genetic disorders[1] to severe combined immunode-
ficiency[2] and cancer.[3] In this regard, original research
used viral carriers to deliver the therapy gene to the tar-
geting cells due to their high transfection efficiency,[4]
However, nonviral gene delivery also faces a set of bar-
riers. One of the challenges is the possible degradation
of the genes by endonucleases in the physiological fluids
and extracellular environment.[8] It has been found that
the half-life of plasmid deoxyribonucleic acid (pDNA)
is only several minutes by an intravenous injection of
gene into mice.[9] Hence, encapsulation pDNA in a robust
nanoparticulate vector is desirable to protect them from
degradation by serum endonucleases and increase cir-
culation time.[8] For example, mixing cationic lipids and
DNA can spontaneously assemble into liposomes or
lipoplexes.[10] Similarly, cationic polymers can condense
T. Wu, Dr. L. Wang, Prof. Y. You
CAS Key Laboratory of Soft Matter Chemistry
Department of Polymer Science and Engineering
University of Science and Technology of China
Hefei, Anhui 230026, China
E-mail: yzyou@ustc.edu.cn
Dr. S. Ding
Department of Pediatrics
The First Affiliated Hospital of Anhui Medical University
Hefei, Anhui 230022, China
E-mail: dsg5312@163.com
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negatively charged DNA through electrostatic interaction
and form condensed complexes (polyplexes).[11] How-
ever, electrostatic interaction among positively charged
polyplexes is highly decreased in the presence of salts,
thereby resulting in colloidal instability. Lipoplexes or
polyplexes tend to break apart or aggregate in biological
fluids which contain serum components and salts.[12]
In particular, aggregation of polyplexes in the blood
can lead to rapid clearance by circulating macrophages
during systemic delivery and inhibit localization to the
desired tissues. Moreover, it may cause embolism in lung
capillaries.[13]
Poly(ethylene glycol) (PEG)-shielding has become
a leading functional strategy to improve stability of
polyplexes in physiological fluids. PEG polymers have
been approved by the Food and Drug Administration
for clinical use due to their low toxicity and no mim-
munogenicity.[14] PEG can be conjugated with cati-
onic polymers to form the outer corona, the resulting
steric stabilization can prevent protein absorption and
lengthen circulation time.[15] PEGylation also reduces
opsonization and complement activation.[16] The hydro-
philicity and phagocytosis of PEGylated polyplexes are
affected by many parameters, such as density of PEG
on the particle surface, the chain length and shape.[14]
According to Gref et al. findings, the molecular weight
between 2 and 5 kDa is optimal for reducing protein
adsorption.[17] PEGylation, however, can cause the
steric hindrance which leads to weaker binding and
influences cellular uptake, decreasing the transfection
efficiency.[18]
In recent years, fluoroalkyl compounds have gained
extensive attention due to their unique properties,
including bioinertness, thermal, and chemical stability,
high rigidity, hydrophobicity, and lipophobicity.[19]
Fluorination can enhance the affinity of polymers to
cell membrane[19a] and the endosome/lysosome mem-
brane.[20] Therefore, fluorination is beneficial to cellular
uptake and endosomal escape of the polymers.[21] More-
over, fluorous compounds are reported to have low toxi-
city and good biocompatibility.[22]
Here, we report a new polypeptide (PEG-PLL-PCys) con-
jugated with fluorocarbon chains. As shown in Scheme 1,
PLL segment can condense plasmid DNA through an elec-
trostatic force to form a complex core, PEG segments sur-
rounding the complex like a corona prevent the complex
from precipitation and reduce the adsorption of serum,
PCys segment with fluorocarbon chains not only improve
the cellular uptake, but also enhance the stability of the
consequent polyplex micelles in physiological condi-
tions. The transfection results show that the fluorinated
polypeptides have remarkably serum resistance and good
gene transfection efficiency even in the presence of 50%
fetal bovine serum (FBS).
Scheme 1. Schematic illustration of the formation of polyplex
micelles and intracellular plasmid DNA delivery.
2. Experimental Section
2.1. Materials
Methoxypolyethylene glycol amine (mPEG-NH2) (Mw is 2 kDa,
Aladdin), N,S-dicarbobenzoxy-l-cysteine (Z-Cys-Z) (Macklin),
N6-(carbobenzyloxy)- l-lysine (Z-Lys-OH) (Adamas), triphosgene
(>98%, Energy Chemical), phosphorus pentachloride (>98%,
Aladdin), pyridine disulfide (>98%, TCI), 2-mercaptoethylamine
hydrochloride (98%, Sigma), pentadecafluorooctanoyl chloride
(>97%, Sigma), perfluorooctanoic acid (>97%, Sigma), were used
as received. N, N′-Dimethylformamide (DMF), tetrahydrofuran
(THF), and toluene were distilled before use.
2.2. Synthesis of N6-carbobenzyloxy-l-lysine N-carboxy-
anhydride (Lys(Z)-NCA)
Lys(Z)-NCA was synthesized using the Fuchs–Farthing method.[23]
In brief, Lys(Z) (4.0 g, 14.2 mmol) was dispersed in dried THF
(60 mL) at 50 °C. To this solution, triphosgene (2.0 g, 6.8 mmol)
was then added. After 3 h, the solution was concentrated and
precipitated into n-hexane, Lys(Z)-NCA was recrystallized from
THF/n-hexane (7/9, v/v).
2.3. Synthesis of S-benzyloxycarbonyl-l-cysteine N-
carboxyanhydride (Cys(Z)-NCA)
Cys(Z)-NCA was synthesized according to the reported litera-
ture.[24] In brief, to an ice-cooled solution of N,S-dibenzyloxycar-
bonyl-l-cysteine (Z-Cys-Z) (2.3 mg, 6.0 mmol) in toluene (25 mL),
phosphorus pentachloride (1.6 g, 7.8 mmol) was added, and the
reaction mixture was shaken for 40 min. Then the clear solution
was heated to 50 °C for 1 h. The anhydride formed was precipi-
tated with excess n-hexane.
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2.4. Synthesis of Poly(ethylene glycol)-b-poly(N6-car-
bobenzyloxy-l-lysine)-b-poly (S-carbobenzoxy-l-cysteine)
(PEG-PLL(Z)-PCys(Z))
DNA polyplexes at different N/P ratios were characterized using
dynamic light scattering (Malvern Zetasizer Nano ZS90). The
morphology structures of the formed polyplexes were charac-
terized using a JEM-2100F transmission electron microscopy
(TEM, 100 KV).
PEG-PLL(Z)-PCys(Z) triblock copolymers were synthesized via
sequential ring opening polymerization of Lys(Z)-NCA and Cys(Z)-
NCA initiated by mPEG-NH2 in anhydrous DMF. mPEG-NH2
(0.2 g, 0.1 mmol) and Lys(Z)-NCA (1.5 g, 5.0 mmol) were dissolved
in 8 mL anhydrous DMF and the reaction was kept for 3 d at 40 °C
under stirring in the presence of N2. Then the solution of Cys(Z)-
NCA (0.8 mg, 3.0 mmol) in 5 mL anhydrous DMF was added, and
the solution was kept for another 3 d at the same condition. The
resulting mixture was precipitated twice in excessive diethyl
ether and the product was dried under vacuum. The final product
has ethylene glycol (EG) units of 44, Lys (Z) of 45, and Cys (Z) units
of 13 calculated from its 1H NMR spectrum.
2.9. Agarose Gel Electrophoresis Assay
Various cationic polymers (PEG-PLL-PCys, PEG-PLL-PCys-SS-3C7F15
,
and PEG-PLL-PCys-SS-5C7F15) were mixed with DNA at different
nitrogen/phosphorus (N/P) ratios for 30 min. The polyplexes
were electrophoresed onto a 0.9% agarose gels and subjected to
electrophoresis for 60 min at 100 V in TAE (Tris base, acetic acid
and EDTA) buffer. Then the gel was soaked in 0.5 µg mL−1 eth-
idium bromide for 30 min and DNA bands were visualized by a
UVP ED3 Imaging System.
2.5. Deprotection of PEG-PLL(Z)-PCys(Z) Triblock
Copolymers
2.10. Confocal Imaging
HeLa cells (10 000 cells per well) in 4-chamber glass dish were
incubated overnight before experiment. The cells were treated
with fluorinated polypeptides/DNA, PEG-PLL-PCys/DNA, PEI/
DNA, and Lipofectamine 2000/DNA polyplexes (DNA labeled
with Cy5), respectively. After incubating for 4 h, the nuclei were
stained with DAPI for 20 min. Then the images were obtained by
confocal laser scanning microscopy (Leica).
Triblock copolymer PEG-PLL(Z)-PCys(Z) (1.0 g) was dissolved in
CF3COOH (8 mL), subsequently, a 33% solution of HBr in HAc
(0.5 mL) was added. After stirring the reaction for 3 h at 0 °C, the
solution was added to excessive diethyl ether and the product
was dried by vacuum.
2.6. Synthesis of N-(2-(2-pyridyldithio)ethyl)
perfluorooctanamide
2.11. In Vitro Gene Transfection
2-(2-Pyridyldithio)ethylamine
hydrochloride
(801.9
mg,
Luciferase pDNA transfection efficiency was measured as fol-
lows: 293T cells were seeded into 96-well plates with the density
of 8 × 103 cells per well in 100 µL of Dulbecco’s modified eagle
medium (DMEM) containing 10% FBS and incubated for 24 h
before transfection experiment. The polyplexes (0.5 µg lucif-
erase plasmid DNA per well) were prepared as follows: PEG-
PLL-PCys-SS-3C7F15 and PEG-PLL-PCys-SS-5C7F15 were complexed
with pDNA at N/P ratio of 30 for 30 min, PEI (N/P 10) and Lipo-
fectamine 2000 (according to the product’s protocols (Invitrogen))
were used as control. Then the polyplexes solution were added
to each well containing 100 × 10−6 m chloroquine and 100 µL of
DMEM with 0%, 10%, 30%, and 50% FBS, respectively. After 4 h
of incubation, the media were replaced with 200 µL fresh DMEM
containing 10% FBS and the cells were cultured for another 48 h.
Then the culture media were removed and cells were washed
with phosphate buffer saline (PBS) buffer (pH 7.4) twice and then
the cells were lysed with 100 µL of cell lysis buffer (Beyotime
Biotechnology) overnight at −80 °C. Subsequently 30 µL thawing
cell lysate was mixed with 50 µL of luciferase substrate for the
determination of relative light units (RLU) using a luminometer
(Thermo fisher, USA). The protein concentration in the cell lysate
was measured by a bicinchoninic acid (BCA) assay kit (Pierce).
The luciferase transfection results are expressed as mean RLU per
milligram of cell protein of protein SD of triplicate experiments.
Green fluorescent protein (GFP) pDNA transfection efficiency
was measured as follows: 293T cells were seeded into 96-well
plates with the density of 8 × 103 cells per well in 100 µL of
DMEM containing 10% FBS and incubated for 24 h before trans-
fection experiment. The polyplexes (0.5 µg luciferase plasmid
3.6 mmol) was dispersed in CH2Cl2 of 40 mL and cooled to −10 °C.
Triethylamine (727.5 mg, 3.6 mmol) was added and stirred for
30 min. Pentadecafluorooctanoyl chloride (1.3 g, 3.0 mmol) dis-
solved in CH2Cl2 (10 mL) was added dropwise within 40 min at
room temperature. After 3 h, the solution was washed with water
twice. The organic layer was dried with anhydrous Na2SO4 and
the volatiles were removed under vacuum. The crude product
was purified by column chromatography (silica gel, hexane/
dichloromethane = 3/1, v/v) to give the product as a colorless
powder (1.7 g, yield: 80%).
2.7. Synthesis of Fluorinated Polypeptides
The fluorinated polypeptides were synthesized as follows. Tri-
block copolymer PEG-PLL-PCys (Mn is 10 kDa) was dissolved in
anhydrous DMF, and N-(2-(2-pyridyldithio)ethyl)perfluorooctana-
mide was added at different molar ratios. The reaction mixtures
were kept stirring for 4 h at N2 atmosphere at room temperature
and then precipitated in diethyl ether, forming PEG-PLL-PCys-SS-
3C7F15 and PEG-PLL-PCys-SS-5C7F15, respectively.
2.8. Characterization of Polymers and Polyplexes
The synthetic polymers were characterized by 1H NMR and
19F NMR (Supporting Information). All NMR were performed on
a Bruker AV 400 NMR spectrometer using chloroform-d, dime-
thyl sulfoxide-d6 (DMSO-d6), and deuterium oxide as the sol-
vent. Zeta potential and size of the fluorinated polypeptides/
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DNA per well) were prepared as follows: PEG-PLL-PCys-SS-3C7F15
and PEG-PLL-PCys-SS-5C7F15 were complexed with pDNA at
N/P ratio of 30 for 30 min, PEI (N/P 10) and Lipofectamine 2000
(according to the product’s protocols (Invitrogen)) were used as
control. Then the polyplexes solution were added to each well
containing 100 × 10−6 m chloroquine and 100 µL of DMEM with
0%, 10%, 30%, and 50% FBS, respectively. After 4 h of incubation,
the media were replaced with 200 µL fresh DMEM containing
10% FBS and the cells were cultured for another 48 h. The expres-
sion of GFP plasmid in the cells was directly observed by a fluo-
rescent microscopy (Olympus), and the transfection efficiency
was quantitatively measured using flow cytometry.
is the most active synthetic method for preparation
of polypeptides,[25] and triblock copolypeptide PEG-
PLL(Z)-PCys(Z) was synthesized by this method via
the successive ring-opening polymerization of Lys(Z)-
NCA and Cys(Z)-NCA initiated by mPEG-NH2. Subse-
quently, a 33% solution of HBr in acetic acid was added
to deprotect benzyloxycarbonyl (Z) groups of the PLL(Z)
and PCys(Z) block, giving PEG-PLL-PCys. The resulting
triblock copolypeptide has a block of 45 Lys units and
a block of 13 Cys units which were calculated from its
1H NMR spectra (Figures S3 and S4, Supporting Infor-
mation). Subsequently, the resulting polypeptide was
partially conjugated with fluorocarbon chains via
disulfide exchange reaction with N-(2-(2-pyridyldithio)
ethyl)perfluorooctanamide as shown in Scheme 2,
resulting in PEG-PLL-PCys-SS-3C7F15 and PEG-PLL-PCys-
SS-5C7F15 (3 and 5 fluorocarbon chains were grafted
onto PCys block, respectively). We measured the 19F
NMR spectra of PEG-PLL-PCys-SS-3C7F15 and PEG-PLL-
PCys-SS-5C7F15 with CF3COOH as an external standard
as shown in Figures S9 and S10 (Supporting Informa-
tion). It can be found that 3 and 5 fluorocarbon chains
were attached to the polypeptides, respectively. We
also measured the UV absorption spectroscopy of PEG-
PLL-PCys, N-(2-(2-pyridyldithio) ethyl) perfluorooc-
tanamid (pyridyldithio-C7F15) and the corresponding
solution after the formation of PEG-PLL-PCys-SS-3C7F15
and PEG-PLL-PCys-SS-5C7F15 (Figure S11, Supporting
Information). Pyridothione produced in the reaction
of PEG-PLL-PCys with pyridyldithio-C7F15 has a strong
absorption at wavelength of 380 nm. All the results
showed that fluorinated polypeptides had been suc-
cessfully synthesized.
2.12. Cytotoxicity of Fluorinated Polypeptides
Cytotoxicity of fluorinated polypeptides was evaluated by MTT
assay. Generally, 293T cells or MCF-7 cells were seeded into
96-well plates with the density of 1 × 104 cells per well in 100 µL
of DMEM containing 10% FBS overnight. The cells were incubated
with fluorinated polypeptides, PEI and Lipofectamine 2000 at dif-
ferent concentrations for 48 h. Subsequently, the culture media
were removed and 20 µL of 5 mg mL−1 MTT stock solution with
a mixture of 100 mL DMEM was added to each well. The yielded
formazan crystals were dissolved in 150 mL DMSO. Absorbance
of the solution in each well was measured at wavelengths of 490
and 570 nm by a microplate reader.
3. Results and Discussion
3.1. Synthesis and Characterization of Fluorinated
Polypeptides
The ring-opening polymerization of amino acid N-car-
boxyanhydrides initiated by primary amine group
O
H
O
O
O
DMF
O
N
O
+
O
N
H
H
45
44
NH
44
2
N
H
N
H
O
HN
O
O
H
N
O
SH
S
O
O
S
O
O
O
H
N
O
O
HBr/HAc
H
13
O
N
H
N
H
45
44
O
H
N
H
O
O
N
H
N
H
DMF
CF3COOH
44
13
45
O
NH2
HN
O
F
F F
F
F
F F
H
N
F
F
N
O
S
S
SH
N
F
O
F F
F F
O
F
O
H
H
N
N
O
N
44
45
H
5
8
H
H
DMF
O
S
S
NH2
HN
F
F
F
O
F
F
F
F
F
F
F
F
F
F
F
F
Scheme 2. Synthesis route of the fluorinated polypeptide.
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3.2. Characterization of Fluorinated Polypeptides
Polyplexes
with 5 fluorocarbon chains have a lower N/P ratio than
that with 3 fluorocarbon chains to completely inhibit
DNA migration demonstrating the introduction of fluo-
rocarbon chains can enhance DNA binding ability.
The fluorinated triblock copolypeptides can condense
DNA via electrostatic interaction, the pKa of PLL is
about 9.4, therefore all primary amino groups of PLL are
protonated in the neutral condition.[26] PLL block can
condense DNA and form a complex core. PEG is hydro-
philic and formed an outer corona, PCys segment with
fluorocarbon can strengthen the binding capacity. The
micelle morphology was characterized by TEM. From
Figure 1A, it can be found that the polyplex micelles
at N/P ratio of 10 is ≈80 nm, which is proper for the
effective endocytosis. The zeta potential values of the
formed polyplexes were demonstrated in Figure 1B at
varying N/P ratios. The zeta potential transformed to
positive value at the N/P ratio of 2, which demonstrated
that the fluorinated polypeptides can bind with DNA at
a low N/P ratio. The positive value increased with the
increase of N/P ratios, and it was 40 mV at the N/P ratio
of 30.
3.4. Stability in the Presence of Heparin and Salts
Generally, the polyplexes are unstable in biological fluids
which contain salts and serum components, in which the
polyplexes tend to break apart or precipitate.[20] In this
work, the stability of fluorinated polypeptides was investi-
gated by agarose gel electrophoresis assay in the presence
of heparin and salts, respectively. As shown in Figure 2A,
at a heparin concentration of 100 µg mL−1, bands corre-
sponding to free DNA were clearly observed for the PEI/
DNA polyplexes, while almost no free DNA was detected
for PLL-PCys-SS-3C7F15/DNA and PEG-PLL-PCys-SS-5C7F15
/
DNA polyplexes. This result demonstrates the substan-
tial stability of fluorinated polypeptides polyplexes in the
presence of heparin. Moreover, the formed fluorinated
polypeptides/DNA polyplexes were stable in the presence
of NaCl. As shown in Figure 2C, the size of PEG-PLL-PCys-
SS-5C7F15/DNA polyplexes micelles remained stable when
treated with 200 × 10−3 m NaCl. The excellent stability of
fluorinated polypeptides/DNA polyplex micelles ascribes
to that the high rigidity of fluorocarbon chains leads to a
low surface energy in physiological fluids.[28] In addition,
the steric stabilization of PEG and potential crosslinking of
PCys segments also played roles in strengthening the sta-
bility of the polyplexes (as shown in Figure S12, Supporting
Information).[29]
3.3. DNA Binding Capacity
In 1975, Laemmli first demonstrated the unusual capa-
bility of polylysine to condense DNA.[27] The DNA-
binding ability of the fluorinated polypeptides was
examined by agarose gel electrophoresis assay. As
shown in Figure 2C, it was very clear that the fluorinated
polypeptides were more efficient in DNA retardation
compared with common polypeptides. The polypeptides
3.5. In Vitro Gene Transfection and
the Serum Resistance of Fluorinated
Polypeptides Polyplexes
Cationic lipids and polymers used in
vivo gene delivery must have a high
stability to tolerate harsh conditions in
biological fluids. Various blood compo-
nents can decrease gene transfection
efficiency by changing the properties
of the polyplexes and dissociating
the polyplexes.[30] In this work, the
fluorinated polypeptides can condense
pDNA well and form robust polyplex
micelles which are stable in physiolo-
gical fluids. The transfection efficiency
and serum resistance of the fluori-
Figure 1. A) TEM image of the polyplexes formed from PEG-PLL-PCys-SS-5C7F15/DNA
at the N/P ratio of 10. B) Zeta potential of the polyplexes formed from PEG-PLL-PCys-
SS-3C7F15/DNA and PEG-PLL-PCys-SS-5C7F15/DNA at different N/P ratios. C) Agarose gel
electrophoresis assay of PEG-PLL-PCys/DNA, PEG-PLL-PCys-SS-3C7F15/DNA, and PEG-PLL-
PCys-SS-5C7F15/DNA polyplexes. D) Agarose gel electrophoresis assay of PLL-PCys-SS-
5C7F15/DNA polyplexes treated with 0 or 20 × 10−3 m glutathione (GSH).
nated polypeptides were assessed in
293T cells with a medium containing
0%, 10%, 30%, and 50% FBS. Chloro-
quine (100 × 10−3 m) was employed as
an ensomolytic agent. As shown in
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Figure 3, the transfection efficiency
of PEG-PLL-PCys-SS-5C7F15/DNA poly-
plexes changed very little with the
increase of serum content and exhib-
ited good GFP transfection efficiency
(35%) even in the presence of 50% FBS
(Figure S16, Supporting Information).
In contrast, gene transfection effi-
ciency of Lipofectamine 2000 and PEI
were significantly decreased in the
presence of FBS. The luciferase trans-
fection efficiency of PEG-PLL-PCys-SS-
5C7F15/DNA polyplexes was about ten
times as that of Lipofectamine 2000
in the presence of 50% FBS. Also, the
polypeptides with
5
fluorocarbon
chains had much higher transfec-
tion efficiency than that with 3 fluo-
rocarbon chains. The polypeptides
with fluorocarbon chains showed
better transfection efficiency than
that without fluorocarbon chains in
the presence of serums. These results
strongly indicate the importance of
fluorocarbon chains in the stabili-
zation of the resulting micelles in
physiological fluids and enhancement
of the gene transfection efficiency in
medium containing FBS.
Figure 2. A) Agarose gel electrophoresis assay of PLL-PCys-SS-3C7F15/DNA, PEG-PLL-PCys-
SS-5C7F15/DNA, and PEI/DNA polyplexes at the N/P ratio of 10 with or without heparin
(HP) at a HP concentration of 100 µg mL−1. B) Agarose gel electrophoresis assay of PEG-
PLL-PCys-SS-5C7F15/DNA polyplexes with 0 m and 200 × 10−3 m NaCl. C) Size stability of
PEG-PLL-PCys-SS-5C7F15/DNA polyplexes in NaCl solution at an N/P ratio of 5.
3.6. Transfection and Serum-Resist-
ance Mechanisms of Fluorinated
Polypeptides
Cellular uptake and DNA release play
important roles in efficient gene
transfection. To explore the mecha-
nisms of the high transfection effi-
ciency of fluorinated polypeptides in
high levels of serum, we compared
the cellular uptake of PEG-PLL-PCys/
DNA polyplexes with PEG-PLL-PCys-
SS-3C7F15/DNA and PEG-PLL-PCys-SS-
5C7F15/DNA polyplexes in HeLa cells
by confocal laser scanning microscopy.
From Figure S14 (Supporting Informa-
tion), it can be found that fluorina-
tion remarkably increased the cellular
uptake efficiency of Cy5-labeled DNA.
And we also compared the cellular
Figure 3. A,B) GFP expressions and luciferase transfection efficiency of PEG-PLL-PCys-
SS-3C7F15/DNA and PEG-PLL-PCys-SS-5C7F15/DNA polyplexes at N/P ratio of 30 in 293T
cells in mediums containing 30% and 50% FBS. PEI (N/P = 10) and Lipofectamine 2000
(according to the product’s protocols (Invitrogen)) are used as control. C) Luciferase
transfection efficiency of PEG-PLL-PCys-SS-5C7F15 at N/P ratio of 30 in 293T cells in
mediums containing 0%, 10%, 30%, and 50% FBS, respectively.
uptake of PEG-PLL-PCys-SS-5C7F15
/
DNA with PEG-PLL-PCys/DNA, PEI/
DNA and Lipofectamine 2000/DNA
polyplexes in the presence of 50%
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Fluorinated PEG-Polypeptide Polyplex Micelles Have Good Serum-Resistance and Low Cytotoxicity for Gene Delivery
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Figure 4. Confocal laser scanning microscopy images of HeLa cells
treated with PEG-PLL-PCys/DNA polyplexes, PEG-PLL-PCys-SS-
5C7F15/DNA polyplexes, PEI/DNA polyplexes, and Lipofectamine
2000/DNA polyplexes in the presence of 50% FBS (DNA was
labeled with Cy5 and the nucleus were stained with DAPI solu-
tion for 20 min).
FBS. As shown in Figure 4, PEG-PLL-PCys-SS-5C7F15
/
DNA polyplexes exhibited much higher cellular uptake
efficiency than those of Lipofectamine 2000/DNA and
PEI/DNA polyplexes which is attributed to the high
stability of fluorinated polypeptides/DNA polyplexes
in physiological fluids. In this work, the fluorocarbon
chains were grafted to the polypeptides by disulfide
bonds. It is well-known that the disulfide bonds can
be reduced after cell internalization since the intracel-
lular environment is much more reductive. As shown in
Figure 1D, when treated with 20 × 10−3 m GSH, bands
corresponding to free DNA were clearly observed for
the PEG-PLL-PCys-SS-5C7F15/DNA polyplexes which can
lead to DNA release.
Figure 5. A,B) Cell viability of PEG-PLL-PCys, PEG-PLL-PCys-SS-
3C7F15, PEG-PLL-PCys-SS-5C7F15, Lipofectamine 2000, and PEI solu-
tion in 293T cells and MCF-7 cells.
3.7. In Vitro Cytotoxicity of Fluorinated Polypeptides
cytotoxicity, and the cell viability dropped to about 30%
above the concentration of 200 µg mL−1. In contrast,
the cytotoxicity of fluorinated polypeptides is low in
The cytotoxicity of gene carriers is a very important
factor for its further application. Synthetic polypeptides
are biocompatible and biodegradable polymers linked
by peptide bond, their structures are similar to natural
proteins.[31] Fluorous compounds are also reported to
have a relative low toxicity and good biocompatibility.
Therefore, we predicted the fluorinated polypeptides
may have a low cytotoxicity. In vitro cytotoxicity was
examined by MTT method in 293T cells and MCF-7 cells
for these fluorinated polypeptides along with Lipo-
fectamine 2000 and commercial PEI-25kDa as control
(Figure 5). PEI showed a quite strong dose-dependent
the entire concentration range from 5 to 200 µg mL−1
.
We also measured the cytotoxicity of PEG-PLL-PCys-SS-
5C7F15 and PEG-PLL-PCys/perfluorooctanoic acid mixture
to simulate the cytotoxicity caused by detachment of
fluorocarbon chains in the cells. As shown in Figure S17
(Supporting Information), both PEG-PLL-PCys-SS-5C7F15
and PEG-PLL-PCys/perfluorooctanoic acid mixture are
nearly nontoxic on the cells. All these results indicate a
very low cytotoxicity of the fluorinated polypeptides in
gene delivery.
Macromol. Biosci. 2017, DOI: 10.1002/mabi.201700114
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Macromolecular
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T. Wu et al.
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4. Conclusions
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sized. PLL segment can condense plasmid DNA through
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complex from precipitation and reduce the adsorption of
serum, while PCys segment with fluorocarbon can enhance
the binding capacity and lead to a significant increase
in the stability of the resulting micelles in physiological
fluids. The fluorinated polypeptides have a low cytotox-
icity and show remarkably serum-resistance, they exhibit
good gene transfection efficiency even in the presence of
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Acknowledgements: This work was supported by the National
Natural Science Foundation of China (51625305, 51273187,
21374107, and 21474097) and by the Fundamental Research
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Conflict of Interest: The authors declare no conflict of interest.
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