Self-Assembled BolA-like Amphiphilic Peptides as Viral-Mimetic Gene Vectors for Cancer Cell Targeted Gene Delivery

Article abstract of DOI:10.1002/mabi.201200283

In this study, two types of BolA-like amphiphilic peptides with dual ligands comprising a tumor-targeting moiety of RGD sequence and a cell-penetrating moiety of R8 sequence are designed and synthesized as gene vectors. The BolA-structural peptide carriers can self-assemble into spherical nanoparticles with a hydrophilic core and shell, which are similar to the viral capsid and can bind plasmid DNA in an aqueous medium to form viral-mimetic complexes. It is found that the BolA-like dual ligands system exhibits significantly enhanced gene expression in both HeLa and 293T cell lines, as compared with poly(ethylenimine) PEI. These BolA-like amphiphilic peptides are promising in clinical trials of gene therapy.

Full text of DOI:10.1002/mabi.201200283

Full Paper
Self-Assembled BolA-like Amphiphilic Peptides
as Viral-Mimetic Gene Vectors for Cancer Cell
Targeted Gene Delivery
Jing-Xiao Chen, Xiao-Ding Xu, Shuo Yang, Juan Yang, Ren-Xi Zhuo,
Xian-Zheng Zhang*
In this study, two types of BolA-like amphiphilic peptides with dual ligands comprising a
tumor-targeting moiety of RGD sequence and a cell-penetrating moiety of R8 sequence are
designed and synthesized as gene vectors. The BolA-structural peptide carriers can self-
assemble into spherical nanoparticles with a hydro-
philic core and shell, which are similar to the viral
capsid and can bind plasmid DNA in an aqueous med-
ium to form viral-mimetic complexes. It is found that
the BolA-like dual ligands system exhibits significantly
enhanced gene expression in both HeLa and 293T cell
lines, as compared with poly(ethylenimine) PEI. These
BolA-like amphiphilic peptides are promising in
clinical trials of gene therapy.
1
. Introduction
to possess all the features comprising of: keeping the
plasmid DNA stability and survival in the blood stream,
penetrating the blood vessel wall, exhibiting specific
binding to the target cells or tissues, internalizing into
cells, enhancing DNA escape from endosomes, transporting
gene toward nucleus, and finally, efficiently inducing
Genetherapyispromisingincuringgeneticdiseasescaused
by DNA defects, such as haemophilia, hypertension,
[
1,2]
diabetes, and cancer.
Since the first clinical trial, it
was found that the success of gene therapy significantly
depends on the availability of appropriate gene carriers
which can overcome barriers during the gene delivery
[
6–9]
the plasmid DNA expression.
However, current gene
vectors more or less present shortcomings and it is
still hard to overcome all kinds of hurdles to reach the
therapeutic target.
[
3–5]
process.
In general, the central dogma of gene therapy is
toachievemaximaltherapeuticefficacywithminimalside-
effects. For this reason, a desirable gene vector is supposed
Gene vectors can be generally divided into viral
vectors and non-viral vectors. As the first generation
of gene vectors, viral vectors are still the most widely
used gene carriers in clinical trials owing to their
desirable performance in gene transfection. Nevertheless,
the development and application of viral vectors is
limited due to biosecurity reasons, low gene loading
J.-X. Chen, X.-D. Xu, S. Yang, J. Yang, R.-X. Zhuo, X.-Z. Zhang
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
[
10–12]
capability, and high cost.
In recent years, non-viral
J.-X. Chen
School of Pharmaceutical Science, Jiangnan University, Wuxi
vectors have attracted more research interest due to
their relatively high biosafety and ease of preparation.
2
14122, P. R. China
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84
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wileyonlinelibrary.com
DOI: 10.1002/mabi.201200283

Self-Assembled BolA-like Amphiphilic Peptides as Viral-Mimetic Gene Vectors for . . .
www.mbs-journal.de
In the past few years, liposomes, dendrimers and cationic
polymers have been developed as non-viral vectors to
solve the contradiction between gene transfection
barriers of gene delivery and the gene transfection
efficiency is still deficient unless these vectors work in
[
42,43]
coordination with other agents or assisting force.
[
13–16]
efficacy and biosafety level.
the transfection efficacy, among these materials, poly-
ethylenimine) (PEI), especially the branched PEI with
Taking into account
Therefore, the future development of peptide-based gene
vectors is to take advantage of a combination of various
bioactive peptide sequences to achieve the optimized
(
[
44,45]
Mn of about 25 kDa, performs extraordinary well
property.
in transferring gene to many cell-lines and has been
In the current study, we designed and fabricated two
types of BolA-like peptide amphiphiles, having dual
hydrophilic functional peptide ligands (tumor-targeting
RGD and cell-penetrating R8 sequences) that combine
with each other via a hydrophobic linkage. The positively
charged R8 sequence is a well-reported cell-penetrating
peptide that can endow the vectors with positive
charges for DNA-binding ability and provide cell-
[
17,18]
considered asthegoldenstandardofnon-viralvectors.
However, PEI is rarely administrated in vivo due to its
low biodegradability and high cytotoxicity. Therefore,
the key issue in recent studies is to develop novel
vectors with high gene delivery capability and good
biocompatibility as well.
Recently, some biomimetic carriers have been reported
for various therapeutic payloads delivery, including
DNA, anti-tumor drugs, vaccines, peptides or proteins,
[
46,47]
penetrating function.
Simultaneously, the incor-
poration of tumor-targeting RGD sequence will endow
the vectors with tumor cell recognition and thus
promote the cellular uptake efficiency through recep-
[
19–21]
and small molecules.
These vectors with unique
structures are able to self-assemble into virus-like
particles, which can enhance the delivery efficacy
via mimicking the unique structures, mechanical
[
48]
tor-mediated endocytosis.
As expected, these BolA-
like amphiphilic peptides could bind DNA and self-
assemble into stable viral-like nanoparticles (Scheme 1).
By using tumor-targeting and cell-penetrating functions,
these BolA-like amphiphilic peptide-based vectors can
efficiently deliver DNA into HeLa cells and present
[
22–25]
properties and functions of virus or viral vectors.
Simultaneously, the gene or drug loading capability of
the self-assembled nanoparticles is much higher than
conventional viral vectors. Moreover, it is easy to endow
these vectors with targeting ability through simple
modifications. Taking the BolA-like amphiphilic peptides
for example, they can self-assemble into a variety of
nanostructures including nanocircles, nanotubes, and
spherical nanoparticles that present a similar topology
structure as the unique architecture of viral capsids
[
26,27]
with hydrophilic cores and surfaces.
Combined
with our recent works on novel vectors based on
peptides with specific sequences that exhibit bio-
activity, good biocompatibility, and high efficacy in
[
28–30]
gene delivery,
in this study, we further designed
and prepared self-assembled BolA-like amphiphilic pep-
tides with dual ligands for cancer cell targeted gene
delivery.
In the past few years, many kinds of peptides with
inherent biodegradability have been developed as
[
31–33]
gene or drug carriers.
Some functional peptide
sequences have been proved to have particular physio-
logical functions including promoting tumor recognition
and endocytosis, such as tumor-targeting peptides
Scheme 1. BolA-like amphiphilic peptides bind with plasmid DNA
to self-assemble viral-mimetic nanoparticles which induce high
gene transfection efficiency with low cytotoxicity. The whole
process is showed as follow: (a) BolA-like amphiphilic peptides
[
34–38]
and cell-penetrating peptides (CPP).
However,
most tumor-targeting peptide sequences can not be
used for DNA binding and delivery separately due to
their small size and insufficient cationic charge density.
Although some efficient cell-penetrating peptides
such as the transcription transactivating peptide (Tat)
and octaarginine (R8) can condense and deliver genes
(
RGD-ADDA-R8) with dual ligands system bind with plasmid DNA
to form viral-mimetic complexes. (b) Next, peptide/DNA com-
plexes internalize into cancer cells through the synergism of
receptor and CPP mediated pathway; (c) endocytic complexes
escape from endosome and plasmid DNA is protected from
degradation by peptide vector; (d) after that, plasmid DNA is
released and trafficked into nucleus, (e) then expression into
protein. (f) In addition, peptide carrier is hydrolyzed into residues
and exocytosed from cells.
[
39–41]
into living cells,
only the cell-penetrating function
of these vectors can not efficiently overcome the
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J.-X. Chen, X.-D. Xu, S. Yang, J. Yang, R.-X. Zhuo, X.-Z. Zhang
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1
significantly enhanced gene expression level as compared
with 25 kDa PEI.
H NMR (DMSO-d
alkyl H), 2.10 (2H, ꢁCH
.10 (2H, ꢁCH ꢁ), 4.20 (1H, ꢁCHꢁ), 7.20–7.80 (8H, arom. H), 12.0
1H, ꢁOH, carboxyl H).
6
) of Fmoc-AHX-OH: d ¼ 1.10–1.50 (6H, ꢁCH
2
ꢁ,
2
ꢁ), 2.70 (1H, ꢁNHꢁ), 2.80 (2H, ꢁCH
2
ꢁ),
4
2
(
2. Experimental Section
2
.1. Materials
2.3. Synthesis of Peptides
N-Fluorenyl-9-methoxycarbonyl (Fmoc) protected L-amino acids
Allpeptides, RGD-ADDA-R8(P1),RGD-AHX-R8(P2), RGD-R8(P3)and
R8 (P4) were designed and synthesized manually employing a
standard solid phase synthetic method based on Fmoc chemistry.
Peptide chains were grown on 2-chlorotrityl chloride resin. The
growthofpeptidechainincludedFmocremovalby 20%piperidine/
DMF (v/v) solution and 2 h coupling cycles. The Fmoc-protected
amino acids were dissolved in DMF and activated by using HBTU/
HOBt/DiEA. Fmoc-ADDA-OH and Fmoc-AHX-OH were coupled in
the same way as other a-amino acids. At the end of the synthesis,
the resin was washed with DMF (three times) and DCM (three
times) and then dried under vacuum for 24 h. After that, the
cleavage of the expected peptides and the removal of side chain
protectivegroupsfromtheresinwereperformedbysuspendingthe
resin in a cleavage cocktail containing TFA (83%), phenol (6.3%),
(
Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH and Fmoc-Asp(OtBu)-OH, 2-
ꢁ1
chlorotrityl chloride resin (100–200 mesh, loading: 0.4 mol ꢀ kg
,
0 0
1
% divinylbenzene (DVB)), o-benzotriazole-N,N,N ,N -tetramethyl-
uronium hexafluorophosphate (HBTU), 9-fluorenylmethoxy carbo-
nyl chloride (Fmoc-Cl) and piperidine were purchased from GL
BiochemLtd.(Shanghai,China)andusedasreceived.Trifluoroacetic
acid (TFA), 1,2-ethanedithiol (EDT), and phenol were provided by
Shanghai Reagent Chemical Co., (China) and used as received.
Thioanisole, 12-aminododecanoic acid (ADDA), 6-aminohexanoic
acid (AHX), and PEI (branched, Mw ꢂ 25 kDa, water-free) were
purchased from Sigma-Aldrich (USA) and used without further
purification. JetPEI, a linear PEI, was synthesized and purified
by PolyPlus-transfection. Diisopropylethylamine (DiEA), N,N-
dimethylformamide (DMF) and dichloromethane (DCM) were
provided by Shanghai Reagent Chemical Co. (China) and distilled
prior to use. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal
bovine serum (FBS), penicillin streptomycin, trypsin, 3-[4,5-
2
thioanisole (4.3%), H O (4.3%), and EDT (2.1%) for 2 h at room
temperature. The filtrate was concentrated to a viscous solution by
rotary evaporation and precipitatedin cold diethyl ether. The crude
product was collected and washed by cold diethyl ether for several
times, and then dried under vacuum. Thereafter, the white powder
was dissolved in distilled water and lyophilized. The purity of the
products was examined by high-performance liquid chromato-
graphy (HPLC) with a C18 column and using a linear gradient of
acetonitrile and DI water containing 0.1% TFA. The molecular
weights of peptides P1, P2, P3 and P4 were determined by electro
spray ionization mass spectrometry (ESI-MS, LCQ Advantage,
Finnigan, USA) (Table 1).
dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide
(MTT),
and Dulbecco’s phosphate buffered saline (PBS) were purchased
from Invitrogen Corp. QIAfilter plasmid purification Giga Kit (5)
was purchased from Qiagen (Hilden, Germany). GelRed was
provided by Biotium (CA, USA). Deoxyribonuclease I (DNase I,
ꢁ1
Activity>500KunitzU ꢀ mg )waspurchasedfromSangonBiotech
Shanghai, China). TheMicrobicinchoninicacid(BCA)proteinassay
(
kit was purchased from Pierce and used as received. All other
reagents and solvents were of analytical grade and used without
further purification.
2
.4. Cell Culture and Amplification of Plasmid DNA
2
93T and HeLa cells were incubated in DMEM medium with 10%
2
.2. Synthesis of Fmoc-ADDA-OH and Fmoc-AHX-OH
ꢁ1
FBS and 1% antibiotics (penicillin-streptomycin, 10 000 U ꢀ mL ) at
37 8C in a humidified atmosphere containing 5% CO
The aminogroupsof ADDAand AHXwereprotectedbyFmoc group
2
.
[49]
according to the previous report.
.020 mol) was suspended in a mixed solution of 1,4-dioxane
20 mL)andaqueousNa CO (10%,100 mL)andthencooledinanice
In brief, ADDA (4.31 g,
PGL-3 plasmid used in this study as the luciferase reporter gene
was transformed in Escherichia coli JM109. The plasmid was
amplified in terrific broth media at 37 8C overnight at 250 rpm, and
purified by an EndoFree QiAfilter Plasmid Giga Kit (5). Then the
purified plasmid was dissolved in TE buffer and stored at ꢁ20 8C.
The integrity of plasmid was confirmed by agarose gel electro-
phoresis, the purity and concentration of plasmid was determined
by UV absorbance at 260 and 280 nm respectively.
0
(
2
3
bath. Subsequently, a solution of Fmoc-Cl (5.18 g, 0.020 mol) in 1,4-
dioxane (40 mL) was added with stirring. The mixture was allowed
to stir at room temperature for 6 h. The 1,4-dioxane was then
removed by rotary evaporation and 250 mL distilled water was
added. Thereafter, the aqueous solution was acidified to pH ¼ 3
with concentrated aqueous HCl, and the product (Fmoc-ADDA-OH)
was extracted with ethyl acetate. After drying over anhydrous
4
MgSO , the crude product was collected by removing the solvent.
2
.5. Preparation of Peptide/DNA and PEI/DNA
The purification was carried out by recrystallization from
acetonitrile. The synthesis procedure for Fmoc-AHX-OH is similar
Complexes
to that of Fmoc-ADDA-OH.
ThepeptidesandPEIweredissolvedin0.150 M NaClbufferandultra
pure water (HPLC grade) respectively. The binary complexes at
different weight ratios (w/w) were prepared by mixing peptides
1
H NMR (DMSO-d
6
) of Fmoc-ADDA-OH: d ¼ 1.10–1.50 (18H,
ꢁ, alkyl H), 2.10 (2H, ꢁCH ꢁ), 2.70 (1H, ꢁNHꢁ), 2.90 (2H,
ꢁ), 4.20 (2H, ꢁCH ꢁ), 4.30 (1H, ꢁCHꢁ), 7.20–7.90 (8H, arom.
ꢁ
CH
CH
2
2
ꢁ1
ꢁ
2
2
and PEI solution respectively with 1.0 mL pGL-3 DNA (100 ng ꢀ mL
in 0.040 M Tris-HCl buffer solution) directly beforehand, and then
H), 12.0 (1H, ꢁOH, carboxyl H).
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Self-Assembled BolA-like Amphiphilic Peptides as Viral-Mimetic Gene Vectors for . . .
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Table 1. Molecular weight of peptides measured by ESI-MS.
3R a)
b)
Peptide Sequence
[M R 3H]
Molecular Weight
P1
P2
P3
P4
RGD-ADDA-RRRRRRRR
RGD-AHX-RRRRRRRR
RGD-RRRRRRRR
612.7
570.2
533.6
423.3
1835.2
1708.9
1595.8
1267.5
RRRRRRRR
a)
b)
M þ 3H]3þ values were found in the ESI-MS spectrum. Theoretical molecular weight.
[
diluted to a total volume of 100 mL with 0.150 M NaCl and vortexed
for 5 s. The complexes were incubated at 37 8C for 30 min upon
mixing before being used for further studies.
incubated in 100 mL DMEM containing 10% FBS for 1 d prior to
adding the peptides. After the peptides were added for 2 d, the
medium was replaced with 200 mL of fresh medium. Then 20 mL
ꢁ1
MTTsolution(5mg ꢀ mL inPBSbuffer)wasaddedtoeachwelland
furtherincubatedfor4 h. Thereafter,themediumwasremovedand
1
50 mL DMSO was added. The absorbance was measured at 570 nm
2
.6. Agarose Gel Electrophoresis Assay
using a microplate reader (Bio-Rad, Model 550, USA). The relative
cell viability was calculated as follows. Cell viability
The peptide/DNA binary complexes at varying weight ratios
ranging from 0 to 40 were diluted to a total volume of 7 mL with
(%) ¼ (OD570(samples)/OD570(control)) ꢃ 100, where OD
570(control)
was
0
.150 M NaCl solution, and then the complexes were incubated at
obtainedin theabsenceof peptides and OD
70(samples)
was obtained
5
3
7 8C for 30 min. For investigating the protection ability of peptide
in the presence of peptides. Each value was averaged from four
independent experiments.
to plasmid DNA, the complexes were then incubated with DNase I
ꢁ1
(
1 mL, 0.5 mg ꢀ mL ) at 37 8C for another 30 min. After that, the
complexes were electrophoresed in 0.7% (w/v) agarose gel
containing GelRed and with Tris-acetate (TAE) running buffer at
2
.10. In Vitro Gene Expression
8
0 V for 60 min. DNA was visualized with a UV lamp using a Vilber
Lourmat imaging system (France).
For gene transfection experiment (luciferase expression), pGL-3
plasmid DNA was used to evaluate the transfection efficiency of
peptides, and the branched PEI (25 kDa) was used as the positive
control. The 293T cells and HeLa cells were seeded in 24-well plates
2
.7. Particle Size and z-Potential Measurement
4
at a density of 6 ꢃ 10 cells per well and cultured with 1 mL DMEM
The particle size and zeta potential of binary complexes were
measured at 37 8C using Nano-ZS ZEN3600 (MALVERN Instru-
ments).Thepreparedcomplexesatvariousw/wratioswerediluted
in ultrapure water to 1 mL volume for the size and zeta potential
measurements. The measurements were repeated 6 runs for each
sample respectively, and the data were reported as the average of
readings.
containing 10% FBS for 1 d respectively. The transfection efficiency
of peptide/DNA complexes was evaluated at a fixed w/w ratio
ranging from 10 to 40, and then incubated at 37 8C for 30 min. Then
thecomplexeswereaddedintotheplatewithserum-freeDMEMfor
4 h at 37 8C. After that, the serum-free DMEM was replaced with
fresh DMEM containing 10% FBS and the cells were further
incubated for 2 d. For luciferase assay, the medium was removed
andcellswerewashedbyPBS,thenthecellswerelysedusing200 mL
reporter lysis buffer (Pierce). The relative light units (RLUs) were
measured with a chemiluminometer (Lumat LB9507, EG&G
Berthold, Germany). The total protein was measured according
to a BCA protein assay kit (Pierce) and luciferase activity was
expressed as RLU per mg protein.
2
.8. Transmission Electron Microscopy (TEM)
Thesampleswere prepared by dipping a coppergrid with formavar
film into the peptide/DNA complexes at w/w ratio of 20. A few
minutesafterthedeposition, excesssolution wasblottedawayand
stained with phosphotungstic acid aqueous solution and then
dried in air. Subsequently, the morphology of peptide/DNA
complexes was viewed on a JEM-100CX II instrument operating
at an acceleration voltage of 80 kV.
2
.11. Laser-Scanning Confocal Microscopy (LSCM)
Observation
The 293T and HeLa cells were seeded in 6-well plates respectively,
and then cells were incubated in 1 mL DMEM containing 10% FBS
for 1 d. To trace plasmid DNA distributed in cells, pGL-3 was stained
by green dye YOYO-1 for 15 min at 37 8C. After that, the complexes
of vector/DNA, prepared by mixing peptide and this stained DNA,
were incubated with cells for 4 h. Subsequently, the medium was
removed and the nuclei of cells were stained by blue molecular
2
.9. In Vitro Cytotoxicity Assay
The cytotoxicity was evaluated with 293T and HeLa cells by MTT
assay. Briefly, the 293T and HeLacells were seeded in 96-wellplates
at a density of 6000 cells per well respectively, and then cells were
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probe Hoechst 33258 for another 15 min. After removing the
medium and washing with fresh medium for six times, the cells
were viewed by using a laser-scanning confocal microscope (Nikon
C1-si TE2000, Japan) under excitation at 408 nm for blue molecular
probe and 488 nm for YOYO-1.
form nanoparticles with a closer architecture, leading to an
increased charge density. With the promotion of hydro-
phobic force, P1 and P2 thus present a stronger DNA-
binding ability as compared with P3. Interestingly, despite
these peptides present different DNA binding capability, all
threepeptides P1, P2andP3are abletoprotectplasmid DNA
from hydrolysis by DNase I at different w/w ratios. As
shown in Figure 1, by treating with DNase I, the
neoformative strips can be observed in the positive control
3. Results and Discussion
3
.1. DNA Binding and Protection Ability of Peptides
(
line2, gel2)implyingtheoccurrenceofenzymaticcleavage
The binding and protection capability of peptide to plasmid
DNA is essential for vectors to condense and protect genes
from degradation in the blood stream. The results in
Figure 1 were tested by agarose gel electrophoresis at
various w/w ratios ranging from 0 to 40. Obviously, BolA-
like amphiphilic peptides P1 and P2 are able to retard DNA
efficiently at w/w ratios of 15 and 20 respectively,
indicating that DNA has been fully condensed at these
w/w ratios. However, P3 with the dual ligands sequence
combined with each other via peptide bond could hardly
condense DNA completely at the w/w ratios below 20.
Although the same arginine residues in these peptides
provide positive charge for DNA-binding, these peptides
exhibit different ability to condense plasmid DNA. The two
BolA-like peptides with hydrophobic spacers are prone to
condense DNA at lower w/w ratios, implying that the BolA-
like structure of peptide vectors plays an important role in
charge aggregation and DNA-binding. In comparison with
P3, due to the presence of a hydrophobic linker in the
molecular structure of P1 and P2, they can aggregate and
of naked plasmid DNA. Although these BolA-like peptides
cannot fully condense plasmid DNA to form complexes at a
low w/w ratio of 15, the neoformative strips do not appear
in line 3 and line 4, demonstrating that these BolA-like
peptides can induce effective protection of DNA from
hydrolysis. In addition, even if P3 presents the weakest
DNA-binding capability, it can also protect DNA from
hydrolysis as well. A reasonable speculation can be raised
that the cationic BolA-like peptides are able to bind DNA
and restrict the combination of DNase I to plasmid DNA.
Because the self-assembled peptide/DNA complexes are
formed through a dynamic process, the cationic peptides
can not only inhibit the enzymatic cleavage of bound DNA,
but also free plasmid DNA at a low w/w ratio.
3
.2. Particle Size and z-Potential Measurement
The particle size and z-potential are two important factors
to affect the intracellular trafficking, cellular uptake and
transfection efficiency. In this study, the average hydro-
dynamic sizes of all peptide/DNA com-
plexes at the w/w ratios ranging from 2 to
4
0 were measured. As presented in
Figure 2, the particle size of BolA-like
peptide/DNA complexes decreases with
the increasing w/w ratios from 2 to 20,
implying the formation of viral-mimetic
nanoparticles. At the w/w ratio of 20,
plasmid DNA is fully condensed by BolA-
like peptides to form the P1/DNA and P2/
DNA complexes with the minimum
hydrodynamic sizes of 168 nm and
2
35 nm, respectively. The P3/DNA com-
plexes obtain the minimum size of
74 nm at w/w ratio of 30, which is in
2
agreement with the results of agarose gel
electrophoresis revealed in Figure 1. The
size of P4/DNA complexes presents a
similar trend as that of P3/DNA com-
plexes and the corresponding minimum
hydrodynamic size is 219 nm at the w/w
ratio of 40.
Figure 1. Agarose gel electrophoresis retardation assay of peptides/DNA complexes at
various w/w ratios. Gel 1 (a1, b1 and c1) and gel 2 (a2, b2 and c2) represent the complexes
untreated and treated with DNase I for 30 min before electrophoresis assay, respect-
ively. Line 1 shows the mobility of naked plasmid DNA used as control, line 2 in gel 2
shows the naked plasmid DNA treated with DNase I for 30 min as the positive control. (a)
P1/DNA complexes, (b) P2/DNA complexes, (c) P3/DNA complexes.
Since the cellular membrane is
negatively charged, a positive charge of
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3
.3. TEM Measurements
The morphology of all peptide/DNA complexes at the w/w
ratio of 20 was observed under TEM and the results are
shown in Figure 4. The sizes of BolA-like peptides/DNA
complexes are less than 100 nm, which is much smaller
than that measured by dynamic light scattering (DLS). The
main reason is attributed to the different test conditions.
Namely, the peptide/DNA complexes measured by DLS are
in the hygrometric state while TEM images are collected
from dry conditions that result in the shrinkage of the
complexes. In comparison with the BolA-like amphiphilic
peptides that can bind DNA to form stable spherical
nanoparticles, peptides P3 and P4 could also bind DNA to
form complexes. However, these complexes are not stable
and apt to aggregate (Figure 4c and 4d). All these results
once demonstrate that gene vectors can be endowed with
much stronger DNA-binding ability by altering the
molecular structures of vectors.
Figure 2. Particle size (average diameter) of peptides/DNA com-
plexes measured by DLS at various w/w ratios as specified. Error
bar represent standard deviation of 6 replicates for the test.
complexes is necessary for cellular uptake and success of
transfection. In addition, the absolute value of z-potential
affects the stability of complexes. As revealed in Figure 3,
the z-potential of binary complexes presents an ascent
trend with the increasing of w/w ratios ranging from 2 to
3
.4. Cytotoxicity Study of Peptides
4
0. The positive z-potential indicates that DNA with
negative charge has been shielded by peptide vectors.
The maximum z-potential of the P1/DNA and P2/DNA
complexes is 22 and 17 mV respectively, which is much
higher than that of P3/DNA (11 mV) and P4/DNA (7 mV)
complexes. The maximum z-potential of the BolA peptide
P1/DNA complexes is nearly 2- and 3-folds higher than that
of P3/DNA and P4/DNA complexes, implying that BolA-like
peptides/DNA complexes are much more stable than P3/
DNA and P4/DNA complexes. The results of size and z-
potential indicate that the structure of BolA-like peptides
can efficiently promote the binding and condensation of
plasmid DNA, strongly demonstrating that the introduc-
tion of a hydrophobic linker can endow the BolA-like
peptides with hydrophobic interaction, increasing the
stability of the peptide/DNA complexes.
The cytotoxicity of BolA-like peptides was evaluated
against 293T and HeLa cells by MTT assay, using 25 kDa
PEI as the positive control. As shown in Figure 5, all peptide
based vectors do not exhibit any obvious cytotoxicity
against the cell lines used in this study. The cell viability of
ꢁ
1
P1, P2, P3, P4 and PEI at 200 mg ꢀ L against HeLa cells is
71.7%, 73.8%, 80.2%, 83.1% and 12.8% respectively. With
Figure 3. z-Potential of peptides/DNA binary complexes
measured at appointed w/w ratios ranging from 2 to 40. Error
bar represent standard deviation of 6 replicates for the test.
Figure 4. TEM images of peptides/DNA binary complexes at w/w
ratio of 20. (a) P1/DNA complexes, (b) P2/DNA complexes, (c) P3/
DNA complexes, (d) P4/DNA complexes.
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J.-X. Chen, X.-D. Xu, S. Yang, J. Yang, R.-X. Zhuo, X.-Z. Zhang
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Figure 5. Cytotoxicity induced by peptides P1, P2 and P3 in (a) HeLa
cells and (b) 293T cells, PEI (25 kDa) was used as positive control.
The relative cell viability was compared with the untreated cells.
Error bars represent standard deviation of 4 replicates for the
test.
Figure 6. In vitro luciferase expression level mediated by peptides
P1, P2, P3 and P4 in (a) HeLa cells and (b) 293T cells. JetPEI/DNA
performed at the optimal w/w ratio of 1.33 was used as positive
control. Error bars represent standard deviation of 3 replicates for
the test.
respectto293T cells, thecell viabilityofP1, P2, P3, P4and PEI
at 200 mg ꢀ Lꢁ is 82.7%, 71.6%, 76.7%, 81.7% and 11.1%.
Either HeLa or 293T cells incubated with these peptides can
maintain relatively high cell viability (more than 70%) even
at a high concentration of vectors, indicating the good
biocompatibility of the peptide vectors. It is known that the
cytotoxicity of gene vectors is related to their molecular
weights, surface charges and effects on membranes
structure. Despite the favorable performance in DNA-
binding and cellular uptake, PEI with high positive charge
density may cause membrane disruption and cell inhibi-
tion. Due to the poor biodegradability, long-term toxicity
causedbyPEIshouldnotbeignored. AsforBolA-likepeptide
vectors used in this study, their positive z-potential values
are modest and hardly induce membrane disruption.
Moreover, peptides with inherent biodegradability can
be hydrolyzed by protease and peptidase in cells, which
could further weaken the cytotoxicity to some extent.
As shown in Figure 6, in comparison with other peptide
vectors, P1 can mediate transfection with much higher
transfection level in both HeLa and 293T cells. Although
BolA-like peptide P2 with a short hydrophobic linker
presents lower transfection efficiency than that of P1, the
transfection efficiency with the changing trend in w/w
ratios is the same as that of P1. In HeLa cells, the peak values
of luciferase expression level induced by P1, P2, P3, P4 and
1
8
6
4
PEI are 4.58 ꢃ 10 (w/w 25), 7.33 ꢃ 10 (w/w 25), 7.89 ꢃ 10
4
8
(w/w 5), 8.45 ꢃ 10 (w/w 30) and 7.49 ꢃ 10 RLU per mg of
protein, respectively. With respect to 293T cells, the
transfection efficiency induced by peptide vectors P1, P2,
8
7
P3,P4andPEIwere5.28 ꢃ 10 (w/w25),1.71 ꢃ 10 (w/w25),
5
5
8
2.37 ꢃ 10 (w/w 5), 1.01 ꢃ 10 (w/w 30) and 9.82 ꢃ 10 RLU
per mg of protein, respectively. Obviously, BolA-like
amphiphilic peptide P1 with a hydrophobic linker presents
muchhigherluciferaseexpressionthanhydrophilicpeptide
carrier P3, which has the same functional peptide motif but
in different connections. In addition, the BolA-like peptide
P1 at its optimal w/w ratio can induce much higher gene
expression than that of P4 and our previous reported
3.5. In Vitro Transfection
7
Because the transfection efficiency is generally affected by
cell type and w/w ratio, the luciferase expression efficiency
of peptide/pGL-3 complexes was evaluated in HeLa and
lipopeptide vectors (LP1, 8.98 ꢃ 10 RLU per mg of protein,
7
[28]
LP2, 9.59 ꢃ 10 RLU per mg of protein),
implying that the
unique BolA-like structure of peptide vectors plays an
important role in gene delivery. Thus, we can infer that the
BolA-like amphiphilic peptide with the aliphatic linker
2
93T cells at the w/w ratio range from 10 to 40. JetPEI at its
optimal w/w ratio of 1.33 was used as the positive control.
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Self-Assembled BolA-like Amphiphilic Peptides as Viral-Mimetic Gene Vectors for . . .
www.mbs-journal.de
Figure 7. Internalization of BolA-like amphiphilic peptides/DNA complexes as observed by LSCM. Images show the delivery of (b, f) YOYO-1
(
green) labeled plasmid DNA to nucleus of (a-d) HeLa cells and (e-h) 293T cells. (a, e) Nucleus was stained by probe Hoechst 33258 (blue). (c, g)
The overlay of DNA and nucleus exhibits the localization of plasmid DNA in cells, and (d, h) the bright field shows the morphology of cells.
Scale bar: 25 mm.
between RGD ligand and CPP (R8) is able to bind DNA
efficiently and form more stable nanoparticles with a viral-
mimetic architecture. Besides, the optimal w/w ratio of
BolA-like peptide performed in gene transfection is
relatively low because the peptide/DNA complexes can
entertumorcellsthroughthesynergismofreceptorandCPP
mediated pathway. The overdosed BolA-like peptides may
compete with the complexes to pass through cell mem-
brane and, to some extent, block the uptake pathway and
induce the decreasing of transfection efficiency at last.
v 3 v 5
the effective recognition by integrin a b and a b over-
expressed on tumor cells of HeLa. Besides, octaarginine
sequence can also promote endocytosis by penetrating the
cell membrane. The synergistic effect between two ligands
and the unique architecture of carriers lead to the efficient
trafficking of plasmid DNA into tumor cells.
4
. Conclusion
Two types of biocompatible BolA-like amphiphilic peptides
with tumor-targeting RGD sequence and cell-penetrating
R8 sequence were designed and synthesized. With the
unique BolA-like structure, these peptides could bind DNA
efficientlyandformviral-mimeticnanoparticles. Duetothe
cell-penetrating function, these two BolA-like amphiphilic
peptidesmightbehelpfulforthetraffickingofDNAthrough
cell membranes and into nucleus, resulting in high gene
expression comparable to that of 25 kDa PEI. Moreover,
since the incorporated RGD sequence provides a tumor-
targeting function, the BolA-like amphiphilic peptides
present an obvious increased gene expression level in HeLa
cells, suggesting a promising potential of the BolA-like
amphiphilic peptides for targeted gene therapy.
3
.6. Cellular Uptake of Peptide/DNA Complexes
To further investigate the tumor cell targeting capability of
peptide carriers, LSCM was used to observe the cellular
uptakeofP1/DNAcomplexinHeLaand293Tcelllines. After
incubating the cells with P1/DNA complex for 4 h, the
delivery of YOYO-1 labeled DNA (green) was viewed under
LSCM. As shown in Figure 7, DNA could be trafficked into
two kinds of cells effectively. By staining the nucleus using
blue molecular probe, it is found that a large amount of
plasmid DNAcould gatherto thenucleus, especiallyin HeLa
cells, which is in agreement with the high gene expression
levelinHeLacells. ComparingFigure7bwith7f, theamount
of P1/DNA complexes in HeLa cells is much higher than that
in 293T cells. The difference in the cellular uptake for these
twocelllinesisduetotheintroductionofRGDligand.Dueto
the unique architecture of viral-mimetic nanoparticles,
RGD ligands distributed on the surface of complex facilitate
Acknowledgements: We acknowledge the financial support from
the Ministry of Science and Technology of China (2011CB606202),
National Natural Science Funds for Distinguished Young Scholar
Macromol. Biosci. 2013, 13, 84–92
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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J.-X. Chen, X.-D. Xu, S. Yang, J. Yang, R.-X. Zhuo, X.-Z. Zhang
www.mbs-journal.de
(
51125014) and the Fundamental Research Funds for the Central
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