Intracellular Delivery of Nanoparticles and DNAs by IR9 Cell-penetrating Peptides
Lee H-J (2013) Intracellular Delivery of Nanoparticles and DNAs by IR9 Cell-penetrating Peptides. PLoS
ONE 8(5): e64205. doi:10.1371/journal.pone.0064205
Intracellular Delivery of Nanoparticles and DNAs by IR9 Cell-penetrating Peptides
Betty R. Liu 0
Ji-Sing Liou 0
Yue-Wern Huang 0
Robert S. Aronstam 0
Han-Jung Lee 0
Joseph Najbauer, University of Pecs Medical School, Hungary
0 1 Department of Natural Resources and Environmental Studies, National Dong Hwa University , Hualien, Taiwan , 2 Department of Biological Sciences, Missouri University of Science and Technology , Rolla, Missouri , United States of America
Cell-penetrating peptides (CPPs) comprised of basic amino residues are able to cross cytoplasmic membranes and are able to deliver biologically active molecules inside cells. However, CPP/cargo entrapment in endosome limits biomedical utility as cargoes are destroyed in the acidic environment. In this study, we demonstrate protein transduction of a novel CPP comprised of an INF7 fusion peptide and nona-arginine (designated IR9). IR9 noncovalently interacts with quantum dots (QDs) and DNAs to form stable IR9/QD and IR9/DNA complexes which are capable of entering human A549 cells. Zetapotentials were a better predictor of transduction efficiency than gel shift analysis, emphasizing the importance of electrostatic interactions of CPP/cargo complexes with plasma membranes. Mechanistic studies revealed that IR9, IR9/QD and IR9/DNA complexes may enter cells by endocytosis. Further, IR9, IR9/QD and IR9/DNA complexes were not cytotoxic at concentrations below 30, 5 and 20.1 mM, respectively. Without labor intensive production of fusion proteins from prokaryotes, these results indicate that IR9 could be a safe carrier of genes and drugs in biomedical applications.
Funding: The financial support of this study was provided by the Postdoctoral Fellowship NSC 101-2811-B-259-001 (to Liu BR) and the Grant Number NSC
1012320-B-259-002-MY3 from the National Science Council of Taiwan (to Lee HJ). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The cell membrane is a permeable barrier that protects living
cells from the extracellular environment by controlling the
movement of materials into and out of cells. The cytoplasmic
membrane mediates a wide range of essential processes, including
environmental sensing, nutrient uptake, cellular morphogenesis,
secretion and cell wall biogenesis . The importance of the
plasma membrane is reflected by the fact that most
pharmaceutical drugs target plasma membrane components . Transport of
exogenous molecules across this barrier is complex and is
influenced by phospholipid, glycolipid, cholesterol and protein
composition. Membrane permeability depends on specific
membrane transporters as well as the size and polarity of molecules of
interest. In the absence of specific transporters, the membrane
only allows the movement of small hydrophobic molecules into the
cell . Large hydrophilic drugs and biological macromolecules,
including DNAs, RNAs and proteins, do not cross cell membranes
Cell-penetrating peptides (CPPs, also known as protein
transduction domains) are a group of short peptides capable of
traversing cell membrane and delivering a variety of cargoes into
living cells . They were originally derived from the viral
transactivation of transcription (Tat) protein that is capable of
crossing cell membranes [9,10]. A basic amino acid-rich region of
the truncated Tat protein was identified as the domain responsible
for penetrating cell membranes and accumulating in cell nuclei
. During the last 15 years, more than 100 varieties of CPPs
have been reported , and 843 CPPs are catalogued on the
CPPsite (http://crdd.osdd.net/raghava/cppsite/) . The
essential feature of CPPs is the ability to transport other molecules into
cells. CPPs include amphipathic, hydrophobic and cationic
peptides . CPPs can be classified into three major families:
protein-derived, synthetic and chimeric . For instance, Tat
and penetratin, two of the first CPPs discovered, are
proteinderived . Nona-arginine (R9) and the model amphipathic
peptide do not have any natural parent proteins and belong to the
synthetic family. Members of the chimeric family incorporate
various functional domains of natural proteins, such as Pep-1 and
transportan . Each family can be divided into several
subgroups based on their origin or sequence characteristics.
In recent years, CPPs have been exploited to deliver biologically
active molecules into cells and are one of the most promising tools
in therapeutics . Recently, more than 20 clinical trials are
using CPPs to deliver macromolecular drug conjugates into
patients with various diseases . CPPs are capable of carrying a
wide spectrum of cargo molecules, including many types of
proteins, nucleic acids, peptide nucleic acids, cytotoxic drugs,
inorganic particles and liposomes [4,6,16]. CPPs can deliver
cargoes with sizes up to 200 nm in diameter . Our laboratory
has used arginine-rich CPPs to deliver proteins , DNAs
, RNAs  and nanoparticles  into cells from
various species. The internalization kinetics of CPPs is rapid, with
a first-order rate constant of 0.007 sec21 . CPPs are not toxic
to most cells [3032,35,3741], and the safety of CPPs has been
demonstrated by a metabolic analysis . Recently, a detailed
study further confirmed that CPPs are nontoxic in vitro and
nonimmunogenic in vivo .
Quantum dots (QDs) are inorganic semiconductor nanocrystals
first introduced in the 1980s . QDs have a size-range of 1 to
100 nm and consist of a few hundred to a few thousand atoms
. QDs are attractive alternatives to fluorescent proteins due to
their colloidal nature, wide excitation properties, and narrow,
sizedependent and composition-tunable emission spectral ranges. QD
advantages over traditional fluorescent proteins include
photostability, high fluorescence quantum yields, resistance to
photobleaching and chemical degradation, and high levels of brightness
. Accordingly, QDs are increasingly being used in
biomedical imaging studies, as cellular labels, intracellular sensors,
deep-tissue and tumor targeting and imaging agents, and
sensitizers for photodynamic therapy . However, QDs do
not readily enter cells, and aggregation often occurs before and
after internalization [47,49]. To overcome these limitations, QDs
have been surface-modified by either covalent  or
noncovalent  linkages with CPPs. Though CPP-facilitated
delivery of QDs reduces the nonspecific absorption and side effects
, QDs are still susceptible to entrapment and sequestration by
endosomes or lysosomes in cells.
Transduction enhancers and endosomolytic agents have been
employed to improve CPP transduction efficiency and to
overcome endosomal/lysosomal entrapment [38,5355]. Most
enhancers, such as pyrenebutyrate  and dimethyl sulfoxide
(DMSO) [38,54], either increase the net hydrophobicity of CPPs
or increase membrane permeability, while chloroquine is a
lysosomotropic agent that prevents lysosomal trapping .
Insofar as endocytosis is one of the primary mechanisms of
cellular uptake of CPPs, quick release from endocytic vesicles into
the cytosol is essential to preserve biological activity of the cargoes
[3,68]. Several lysosomotropic peptides, also called
endosomedisruptive peptides or membrane destabilizing peptides, have been
derived from viral and bacterial toxins . These peptides trigger
endosomal acidification that leads to cargo escape into the cytosol
. INF7 peptide, a glutamic acid-enriched influenza virus
hemagglutinin-2 (HA2) analogue, has been shown to be a
particularly potent fusion peptide , as was used in the present
The aims of this study were to (1) create a chimeric IR9 CPP
containing both INF7 fusion peptide and R9 (designated IR9), (2)
evaluate the transduction of IR9 for cellular delivery of QDs and
plasmid DNAs and (3) determine the mechanism of CPP-mediated
uptake of QDs and DNAs in cells. To achieve these goals, we
synthesized IR9 and examined the intracellular delivery of IR9,
IR9/QD and IR9/DNA using live cell imaging and flow
cytometry. To understand the relationship between transduction
efficiency and IR9/cargo ratios, the charging state and
electrostatic interactions of IR9/cargo complexes were characterized
using a zeta-potential analyzer. To elucidate the uptake
mechanisms of IR9 and IR9/cargo complexes, physical and
pharmacological inhibitors were used to block specific endocytic pathways.
Finally, the cytotoxicity of IR9 and IR9/cargo complexes was
Materials and Methods
Human bronchoalveolar carcinoma A549 cells (American Type
Culture Collection, Manassas, VA, USA; CCL-185) were
maintained in Roswell Park Memorial Institute (RPMI) 1640 medium
(Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10%
(v/v) bovine serum (Gibco) . Living cells were determined by
propidium iodide stain. Cells were washed with phosphate
buffered saline (PBS) three times before and after each treatment.
The culture medium was switched to RPMI 1640 medium
supplemented with 1% serum during incubation with IR9-FITC,
IR9/QD or IR9/DNA.
QDs and Preparation of Peptides
Carboxyl-functionalized CdSe/ZnS QDs eFluor 525NC (green
fluorescent QD) and eFluor 625NC (red fluorescent QD; denoted
as QDr) possess the maximal emission peak wavelengths of 525
and 625 nm, respectively (eBioscience, San Diego, CA, USA). IR9
of 92.9% purity was chemically synthesized (Genomics, Taipei,
Taiwan). IR9-FITC peptide of 90.2% purity contained the
fluorescein isothiocyanate (FITC) at the N-terminus (Genomics).
The molecular masses of IR9 and IR9-FITC are 3996.6 and
4499.1 Dalton, respectively.
Gel Retardation Assay
To prepare IR9/QD complexes, IR9 peptide was mixed with
2 mM of QDs at molecular ratios of 0 (QD only), 15, 30, 60, 90
and 120 in PBS, and incubated at 37uC for 2 h. IR9/QD
complexes were analyzed by electrophoresis on a 0.5% agarose
gel (Multi ABgarose, Thermo Fisher Scientific, Waltham, MA,
USA) in 0.5 6 TAE (40 mM of Tris-acetate and 1 mM of
EDTA, pH 8.0) buffer at 100 V for 40 min, as previously
described . To prepare IR9/DNA complexes, different
amounts of IR9 were mixed with 3 mg of the pEGFP-N1
plasmid (Clontech, Mountain View, CA, USA) encoding the
enhanced green fluorescent protein (EGFP) reporter gene at
various molar nitrogen/phosphate (NH3+/PO42 or N/P) ratios
of 0 (DNA only), 0.5, 1, 1.5, 2, 2.5, 3, 6, 9, 12 and 15. After
2 h incubation, the IR9/DNA mixtures were analyzed by
electrophoresis on a 0.5% agarose gel at 100 V for 40 min and
stained with SYBRH Green 1 (Molecular Probes, Eugene, OR,
USA), as previously described . Images were captured using
a Typhoon Trio imager (GE Healthcare, Piscataway, NJ, USA)
with the excitation wavelength of 532 nm (SYAG laser) and
with the emission of 532 nm. Data were analyzed using
ImageQuant TL 7.0 software (GE Healthcare).
Noncovalent QD Transduction
In the protein transduction experiments, different amounts of
IR9-FITC peptide (0, 1, 5, 10, 30 and 60 mM) were incubated
with human A549 cells for 1 h, and cells were then analyzed with
flow cytometry. Cells treated with PBS, FITC (Sigma-Aldrich, St.
Louis, MO, USA) or a non-CPP (casein-FITC, Sigma-Aldrich)
served as negative controls. In the kinetic study of transduction,
5 mM of IR9-FITC were added to cells for 0, 1, 5, 10, 30 and
60 min at 37uC. To determine subcellular colocalization of
IR9FITC, organelle-specific fluorescent trackers Hoechst 33342
(Invitrogen) and LysoTracker DND-99 (Invitrogen) were utilized
to visualize nuclei and lysosomes, respectively, according to the
manufacturers instructions. Cells were treated with 5 mM of
IR9FITC for 1 h and stained with both trackers .
For the transduction of noncovalent IR9/cargo complexes, 1, 5,
10, 30 and 60 mM of IR9 peptide were mixed with QDs at a
molecular ratio of 60 at 37uC for 2 h. IR9/QD complexes were
then incubated with the cells at 37uC for 1 h. To study
transduction kinetics, cells were treated with IR9/QD complexes
prepared at a molecular ratio of 60 for 0, 1, 5, 10, 30, 60 min,
12 h and 24 h at 37uC. In other experiments with high molecular
ratios, cells were treated with IR9/QD complexes prepared at
molecular ratios of 120 and 240 for 24 h followed by analysis with
a confocal microscope or a flow cytometer. To test QD
dissociation from CPPs, 5 mM of IR9-FITC was mixed with
2.6 nM of QDr at 37uC for 2 h, and then incubated with cells for
24 h. Following incubation, IR9-FITC/QDr complexes were
removed and cells were stained with Hoechst 33342, followed by
observation using a confocal microscope. Lysosomal escape was
conducted by adding 25 mM of chloroquine (Sigma-Aldrich) to
cells previously treated with IR9/QD complexes for 24 h. The
Figure 2. Noncovalent interactions between IR9 and QDs in vitro. (A) Gel retardation assay revealing stable interactions between IR9 and QDs.
Different amounts of IR9 were mixed with QDs at molecular ratios of 0 (QD only), 15, 30, 60, 90 and 120. IR9/QD mixtures were subjected to
electrophoresis on a 0.5% agarose gel. Fluorescence of QDs was visualized at 532 nm using a Typhoon Trio imager (GE Healthcare). (B) Relative shift
of IR9/QD complexes formed at different IR9/QD ratios. Data are presented as mean 6 SD from 6 independent experiments in each treatment group.
cells were then stained with LysoTracker DND-99 and Hoechst
To evaluate the role of endocytosis in complex transduction,
physical and pharmacological endocytic modulators were used
. Cells were incubated at 4uC for 30 min to deplete energy
required by all endocytic pathways; IR9-FITC, IR9/QD or IR9/
DNA complexes were then incubated with the cells at 4uC. To
analyze the role of macropinocytosis in complex transduction, cells
were treated with 10 mM of cytochalasin D (CytD), 100 mM of
5(N-ethyl-N-isopropyl)-amiloride (EIPA), 5 mg/ml of filipin or
10 mM of nocodazole (Sigma-Aldrich) for 1 h to block F-actin
rearrangements, macropinocytosis, caveolae-dependent
endocytosis or clathrin-dependent endocytosis, respectively. The cells were
then treated with IR9-FITC, IR9/QD or IR9/DNA complexes
and uptake determined.
DNA Delivery Mediated by IR9 and Functional Gene
To prepare fluorescence-labeled DNAs, the pBlueScript-SK+
plasmid (Agilent Technologies, Santa Clara, CA, USA) was
labeled with the LabelIT Cyanine 3 (Cy3) nucleic acid labeling kit
(Mirus Bio, Madison, WI, USA) . Cells were seeded at a
density of 1 6 104 per well of 96-well plates. Three mg of
Cy3labeled pBlueScript-SK+ plasmid DNA was incubated with IR9 at
N/P ratios of 0 (Cy3-labeled DNA only), 1, 3, 6 and 9 in a final
volume of 100 mL for 2 h at 37uC. The IR9/Cy3-labeled DNA
complexes were added to cells in the 96-well plates, and the plates
were incubated for 1 h at 37uC. The cells were washed three times
with PBS to remove free IR9/Cy3-labeled DNA complexes, and
then staining with Hoechst 33342.
In a functional gene assay, cells were treated with either 3 mg of
the pEGFP-N1 plasmid DNA mixed with IR9 at N/P ratios of 0
(control), 3, 6, 9 or 12. These complexes were transferred to cells in
each well for 1 h at 37uC. The cells were then washed three times
with PBS. The cells were supplemented with 100 mL of 10%
serum-containing medium and incubated at 37uC for 48 h, and
then stained with Hoechst 33342 and observed using a confocal
Figure 4. Confocal microscopy of intracellular delivery of IR/QD complexes into A549 cells. (A) Images of A549 cells treated with IR9/QD
complexes prepared at various combination ratios. IR9 was mixed with QDs at molecular ratios of 120 and 240, and then incubated with cells for 24 h
at 37uC. The cells were stained with Hoechst 33342 and then observed using a BD Pathway 435 System (BD Biosciences) at a magnification of 6006.
GFP and BFP channels revealed the distribution of QDs and nuclei, respectively. Cell morphologies are shown in bright-field images. Overlaps
between QDs and nuclei are cyan in merged GFP and BFP images. Scale bar is 25 mm. (B) Association between IR9 and QDs after cellular
internalization. Cells were treated with IR9-FITC and QDr as controls. Five mM of IR9-FITC was mixed with 2.6 nM of QDr at 37uC for 2 h, and IR9-FITC/
QDr complexes were added to cells for 24 h at 37uC. Cells were stained with Hoechst 33342 and observed using a Leica confocal microscope system
at a magnification of 1,2606. GFP, RFP and BFP channels revealed the distribution of IR9-FITC, QDr and nuclei, respectively. Overlaps between
peptides and QDr were yellow in merged GFP and RFP images. (C) Subcellular colocalization of IR9-delivered QDs. Cells were treated with IR9/QD
complexes prepared at a molecular ratio of 120 for 24 h in the absence or presence of 25 mM chloroquine. Cells were stained with LysoTracker
DND99 and Hoechst 33342, and images were then observed using a BD Pathway System at a magnification of 6006. GFP, RFP and BFP channels displayed
the distribution of QDs, lysosomes and nuclei, respectively. Overlaps between QDs and lysosomes were yellow/orange color in merged GFP and RFP
Fluorescent and Confocal Microscopy
Fluorescent and bright-field images were recorded using a BD
Pathway 435 bioimaging system (BD Biosciences, Franklin Lakes,
NJ, USA) which includes both the fluorescent and confocal
microscopic sets without a pinhole . Excitation filters were at
377/50, 482/35 and 543/22 nm for blue, green and red
fluorescence, respectively. Emission filters were at 435LP
(longpass), 536/40 and 593/40 nm for blue (BFP), green (GFP) and red
fluorescent protein (RFP) channels, respectively. Confocal images
were also obtained using the TCS SP5 II confocal microscope
system (Leica, Wetzlar, Germany). The parameters for this
confocal microscopy were as follows: excitation at 405 nm and
emission at 435480 nm for the detection of BFP; excitation at
488 nm and emission at 495540 nm for the detection of GFP;
and excitation at 543 nm and emission at 590665 nm for the
detection of RFP. Relative intensities of fluorescent images were
quantified using UN-SCAN-IT software (Silk Scientific, Orem,
UT, USA). Bright-field microscopy was used to assess cell
Flow Cytometric Analysis
Cells were seeded at a density of 2.5 6 105 per well of 24-well
plates. Cells in the control and experimental groups treated with
IR9-FITC or IR9/cargo complexes were harvested and counted
using a Cytomics FC500 flow cytometer (Beckman Coulter,
Fullerton, CA, USA) with a FL1 filter for GFP detection [19,24].
Data were analyzed using CXP software (Beckman Coulter).
IR9 (12.4 mM), QD (100 nM), pEGFP-N1 plasmid DNA (7 mg),
IR9/QD complexes formed at molecular ratios of 60, 120 and
240, and IR9/DNA complexes formed at N/P ratios of 9 and 12
were prepared in double deionized water. Each solution was
temperature-equilibrated at 25uC for 120 sec in a zeta cell.
Zetapotentials of samples were measured using a Zetasizer Nano ZS
(Malvern Instruments, Worcestershire, UK) and analyzed using
Zetasizer software 6.30 (Malvern) . The correlation coefficient
analysis between zeta-potential and protein transduction efficiency
was plotted using SigmaPlot software (Systat, Chicago, IL, USA).
Figure 5. Noncovalent interactions between IR9 and plasmid DNAs in vitro. (A) Gel retardation assay of IR9/DNA complexes. Different
amounts of IR9 were mixed with the pPEGFP-N1 plasmid at molecular ratios of 0 (DNA only), 0.5, 1, 1.5, 2, 2.5, 3, 6, 9, 12 and 15, as indicated. After a
2 h incubation, IR9/DNA complexes were analyzed by electrophoresis on a 0.5% agarose gel and stained by SYBRH Green 1. (B) The relative shift
percentage (y-axis) as a function of IR9/DNA ratio.
Cells were treated with 1, 5, 10, 30 and 60 mM of IR9-FITC,
IR9, IR9/QD or IR9-FITC/QDr complexes prepared at CPP/
probe ratios of 60:1, 120:1 and 240:1 for 24 h at 37uC. To
measure the transduction of IR9/DNA complexes, cells were
treated with IR9/pEGFP-N1 complexes prepared at N/P ratios of
0 (DNA only), 1, 3, 6, 9 and 12 for 24 h at 37uC. Cells without any
treatment served as a negative control, while cells treated with
100% DMSO served as a positive control . Cell viability was
determined using the
1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay .
Results are expressed as mean 6 standard deviation (SD). Mean
values and SDs were calculated from at least three independent
experiments of triplicates per treatment group. Comparisons
between the control and treated groups were performed by the
Students t-test using levels of statistical significance of P,0.05 (*,
a, b) and 0.01 (**, aa, bb), as indicated.
To determine the kinetics of protein transduction of the novel
IR9 peptide, human A549 cells were treated with FITC,
caseinFITC, or various amounts of IR9-FITC peptide and analyzed by
flow cytometry. Cellular internalization of IR9-FITC peptide was
concentration-dependent (Fig. 1A). The kinetic experiment
showed that internalization of IR9-FITC is time-dependent,
entering into cells within 1 min and reaching a stationary phase
at 60 min (Fig. 1B). The IR9-FITC was distributed evenly
throughout the cytosol. Merged images indicated that IR9-FITC
is colocalized with lysosomes (Fig. 1C). These results demonstrate
that IR9 possesses protein transduction ability.
Noncovalent interactions between IR9 and QDs were
confirmed in an agarose-based gel retardation assay (Fig. 2A).
Semiquantitative analysis revealed ratio-dependent interactions of IR9/
QD complexes reaching a plateau at a molecular ratio of 60
(Fig. 2B). Accordingly, this ratio was used in subsequent
To study IR9-dependent cellular internalization, cells were
treated with IR9/QD complexes containing different amounts of
IR9 but a fixed ratio of 60 with QDs. The transduction of IR9 was
concentration-dependent (Fig. 3A), and intracellular accumulation
of IR9/QD complexes could be detected within 1 min (Fig. 3B).
We noted stronger fluorescence at a molecular ratio of 60 of IR9/
QD complexes in 24 h compared to that in 12 h (Fig. 3C and D).
A much larger fraction of the cells internalized IR9/QD
complexes at a molecular ratio of 120 compared to that at a
ratio of 60 (Fig. 3C and D). These data suggest that IR9 not only
can interact with QDs to form stable IR9/QD complexes (Fig. 2),
but also can deliver QDs into cells (Fig. 3).
To reveal the subcellular localization of IR9-delivered QDs,
cells were treated with QDs only or IR9/QD complexes at various
combination ratios, and then stained with a nucleus-specific
fluorescent marker Hoechst 33342. The IR9-delivered QDs were
evenly distributed in the cytosol (Fig. 4A). To address whether
CPPs and cargos dissociate after cellular internalization, cells were
treated with IR9-FITC/QDr complexes, and then stained with
Hoechst 33342. The merged image showed that QDs were largely
colocalized with IR9 within cells (Fig. 4B). This indicates that QDs
remain associated with IR9 following cellular entry. Additionally,
lysosomal escape was affected using the lysosomotropic agent
chloroquine. The cells were then stained with nucleus- and
lysosome-specific fluorescent markers (Hoechst 33342 and
LysoTracker DND-99, respectively). The QDs initially colocalized with
lysosomes, and chloroquine facilitated cytoplasmic distribution of
IR9-delivered QDs (Fig. 4C). Green puncta were observed in the
cells treated with IR9/QD complexes, but no green puncta were
seen in the cells treated with IR9/QD complexes and chloroquine
(Fig. 4C). These results suggest that intracellular delivery of IR9/
QD complexes may involve an endocytic pathway, and
chloroquine promotes lysosomal escape.
To assess whether IR9 can deliver genes into cells, we switched
QD cargoes to plasmid DNAs. A gel retardation assay was
conducted with IR9 and the pEGFP-N1 plasmid DNA. Results
indicated that IR9 noncovalently associates with DNAs to form
stable IR9/DNA complexes (Fig. 5A). DNA mobility decreased as
the amount of IR9 increased (Fig. 5B). Thus, IR9 interacts with
DNAs to form stable IR9/DNA complexes in vitro.
To determine whether DNA plasmids are delivered by IR9 into
cells, A549 cells were treated with IR9/Cy3-labeled
pBlueScriptSK+ plasmid DNA complexes at various N/P ratios, stained with
Hoechst 33342, and then observed using a fluorescent microscope.
No signal was detected in cells treated with Cy3-labeled DNA
alone (Fig. 6A). In contrast, red fluorescence was observed in cells
treated with IR9/Cy3-labeled DNA complexes when N/P was
.1. This indicates that IR9 can transport DNA into cells. In the
functional gene assay, cells were treated with IR9/pEGFP-N1
complexes at different N/P ratios and stained with Hoechst 33342.
No signal was detected in cells treated with the pEGFP-N1
plasmid DNA encoding an EGFP reporter gene (Fig. 6B). In
contrast, green fluorescence was observed in cells treated with
IR9/DNA complexes when N/P was .6, indicating that plasmid
DNAs delivered by IR9 can be actively expressed by cells.
To understand the contribution of N/P ratio in transduction
efficiencies of IR9/cargo complexes (i.e., Fig. 2 versus Fig. 3D and
4; and Fig. 5 versus Fig. 6), the charge state of IR9, cargo and IR9/
cargo complexes were characterized using a zeta-potential
analyzer. Zeta values of carboxyl-functionalized QDs and
arginine-rich IR9 were 225.162.2 mV and 32.861.3 mV,
respectively (Fig. 7A). The surface charge of IR9/QD complexes
formed at a molecular ratio of 60 was near neutral (3.761.2 mV).
However, zeta values of IR9/QD complexes were dramatically
elevated at molecular ratios above 60 (Fig. 7B). This relationship
was also observed with IR9/DNA complexes. While plasmid DNA
has a zeta-potential of 237.661.9 mV, the zeta value of IR9/
DNA complexes was 4.9260.5 mV at an N/P ratio of 9 and
32.260.7 mV at an N/P ratio of 12 (Fig. 7C). A logarithmic curve
was plotted with zeta value against protein transduction efficiency
to generate an equation of y = 41.676Ln(x) 235.16 with an
Rsquared value of 0.9737 (Fig. 7D). The correlation coefficient
analysis demonstrated a high correlation between the
zetapotential and transduction efficiency of CPP/DNA complexes.
Thus, in addition to gel shift ability, the electrostatic interactions of
CPP/cargo complexes can be a predictor of transduction
efficiency within the charge range tested.
We used physical and pharmacological inhibitors to elucidate
mechanisms of cellular internalization of IR9 and its associated
cargoes. Cells were treated with PBS (as a negative control),
IR9FITC, IR9/QD or IR9/DNA complexes in the absence or
presence of endocytic inhibitors, followed by flow cytometric
analysis. We found that cellular uptake of IR9-FITC was sensitive
to treatments of 4uC, CytD and EIPA (Fig. 8A). Cellular
internalization of IR9/QD complexes was inhibited by treatments
of 4uC, CytD, EIPA and nocodazole (Fig. 8B). Cellular entry of
IR9/DNA complexes was sensitive to treatments of 4uC, CytD,
EIPA, filipin and nocodazole (Fig. 8C and D). These results
indicate that the classical energy-dependent endocytosis may be
one of the main routes for cellular internalization of IR9 and IR9/
The MTT assay was performed to determine the effect of
IR9mediated cargo delivery on cell viability. Cells were treated with
IR9-FITC, IR9, IR9/QD, IR9-FITC/QDr or IR9/DNA
complexes for 24 h. IR9 alone and IR9/QD complexes were not
cytotoxic at concentrations below 30 mM, while IR9/QD
complexes above 5 mM at a molecular ratio of 240 were toxic
(Fig. 9A). IR9/DNA complexes showed no cytotoxicity when N/P
was ,12, corresponding to concentrations below 20.1 mM
In this study, we introduce and characterize a novel
cellpenetrating peptide without labor intensive production of proteins
from prokaryotes. IR9 is a chimeric molecule derived from fusion
of synthetic nona-arginine and the fusogenic peptide INF7. IR9
can noncovalently interact with QDs and DNAs to form stable
complexes and deliver both into human A549 cells. A high
correlation is noted between zeta-potential and protein
transduction efficiency of CPP/DNA complexes. Electrostatic interactions
of IR9/cargo complexes with the plasma membrane play an
important role in cellular internalization. Endocytosis may be one
of the main routes for cellular uptake of IR9 and IR9/cargo
complexes. Neither IR9 nor IR9/cargo complexes are cytotoxic at
low concentrations. These properties indicate that IR9 may be a
useful tool in the study of biological processes, including gene
expression, as well as a delivery vector in biomedical applications.
Poor intracellular trafficking and endosomal release are major
factors that reduce efficiency of CPP protein transduction
mediated by endocytic pathways [3,6,8,56]; endosomal
entrapment can lead to enzymatic degradation of CPPs and their
cargoes. Entrapment in endosomes or macropinosomes can be
overcome by incorporating HA2 and INF7 peptides into CPPs to
induce perturbation of vesicle membranes [57,6164]. We
recently reported that the endosomolytic HA2 tag increases
cellular uptake, accelerates endosomal escape, and promotes the
even cytosolic distribution of endocytosed CPP-containing RFPs in
human A549 cells . The working concentrations of the
cumbersome bacteria-produced R9-HA2-mCherry  and
synthetic IR9-FITC were 30 and 5 mM, respectively. The relative
transduction efficiency of 5 mM of IR9-FITC was much higher
than that of 5 mM of R9-HA2-mCherry, whereas similar
transduction efficiencies were noticed at high concentrations (both
30 and 60 mM) of R9-HA2-mCherry and IR9-FITC. Less
cytotoxicity was detected with 30 mM of R9-HA2-mCherry;
however, the same concentration of IR9 reduced cell viability.
Membrane potential plays a critical role in the internalization of
arginine-rich CPPs into cells [66,67]. We hypothesized that the
charge state of IR9/cargo complexes can influence the efficiency
of CPP-mediated cellular internalization. Protein transduction of
CPP-mediated cargo delivery can be envisioned as a three-step
process: first, binding to cellular membranes; second, penetration
into cells; and third, release into cytoplasm or specific organelles
[6,58]. The first step of cellular uptake is initiated by electrostatic
interactions between CPP/cargo complexes and negatively
charged plasma membranes . For instance, surface charge is
a major determinant of how gold nanoparticles impact cellular
processes, such as cell morphology, mitochondrial function,
intracellular calcium levels and cytotoxicity . Both positively
and negatively charged gold particles are cytotoxic, with the
negative ones being more toxic. Recently, electropositive
zetapotentials of nanodiamond particles were found to vary greatly
depending on nanoparticle size, methods of production and
treatment, surface structure and other properties [39,69]. In our
gel retardation assay, IR9/QD complexes attained a plateau of
complex formation at a molecular ratio of 60 (Fig. 2), although
higher transduction efficiencies were attained with higher
molecular ratios, such as 120 (Fig. 3D and 4A). The transduction
efficiency of IR9/cargo complexes correlated well with the
magnitude of electropositive zeta-potentials of IR9/cargo
complexes (Fig. 7). Electropositivity at or above 25 mV can be taken as
an arbitrary value separating low-charged surfaces from
highlycharged surfaces that contributes to suspension stability in colloidal
systems . Hence, this electrostatic property that governs CPP/
cargo complex interactions with the negatively charged plasma
membranes appears to be the key factor in determining
transduction efficiency, rather than simple complex formation
per se (as indicated by gel shift assay).
Though the understanding of cellular internalization of CPP/
cargo complexes is still incompletely understood, the general belief
is that most CPPs utilize two or multiple pathways for cellular
entry . Two major routes for cellular uptake of CPPs are the
endocytic and nonendocytic pathways. Classical endocytosis is an
energy-dependent pathway and includes both phagocytosis and
pinocytosis [71,72]. The nonendocytic route (also called direct
membrane translocation, direct penetration, or pore-opening
mechanism) is a rapid and energy-independent pathway [3,73].
Figure 9. Cytotoxicity of IR9 and IR9/cargo complexes. (A) Influence of IR9-FITC, IR9 and IR9/QD complexes on A549 cell viability. Cells were
treated with different concentrations (1, 5, 10, 30 and 60 mM) of IR9-FITC, IR9 or IR9/QD complexes prepared at molecular ratios of 60, 120 and 240, or
IR9-FITC/QDr complexes. After a 24 h incubation at 37uC, mitochondrial succinate dehydrogenase activity was analyzed using the MTT assay. Cells
treated with PBS and DMSO for 24 h served as negative and positive controls, respectively. (B) Influence of DNA and IR9/DNA complexes on cell
viability. Cells were incubated with IR9/pEGFP-N1 complexes prepared at N/P ratios of 0 (DNA only), 1, 3, 6, 9 and 12 at 37uC for 24 h, as indicated.
Cells treated with PBS and DMSO for 24 h served as negative and positive controls, respectively. The MTT assay was used to evaluate cell viability.
Significant differences of P,0.05 (*) and P,0.01 (**) are indicated. Data are presented as mean 6 SD from 3 independent experiments in each
Antennapedia, R9 and Tat have been reported to simultaneously
use at least three endocytic pathways: macropinocytosis,
clathrinmediated endocytosis and caveolae/lipid-raft-mediated
endocytosis . In this study, endocytosis was found to be one of the main
routes for cellular uptake of IR9 and IR9/cargo complexes (Fig. 8).
The pathways involved in the internalization of QDs depend on
their conjugated peptides or carriers . CPP properties,
CPP concentration, cargo characteristics, CPP/cargo complexing
method, duration of transduction, serum concentration and
composition of cell membrane have all been reported to influence
cellular uptake efficiency and pathways of CPPs [56,74,7881].
A novel CPP, IR9, noncovalently interacts with QDs or DNAs
to form stable complexes that are able to deliver into human A549
cells. Electrostatic interactions of IR9/cargo complexes with
cellular membranes play a key role in cellular internalization. IR9
and IR9/cargo complexes may enter cells by endocytosis. IR9
includes the INF7 fusogenic domain which promotes the release of
IR9/cargo complexes from endosomes. IR9 is relatively nontoxic
and may be an excellent carrier of therapeutic cargoes in
We thank Chia-Liang Cheng (Department of Physics, National Dong Hwa
University, Taiwan) for performing the zeta-potential measurements, and
Conceived and designed the experiments: HJL. Performed the
experiments: BRL JSL. Analyzed the data: YWH RSA HJL. Wrote the paper:
HJL BRL YWH RSA.
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