A Cancer Specific Cell-Penetrating Peptide, BR2, for the
Efficient Delivery of an scFv into Cancer Cells
Ki Jung Lim1, Bong Hyun Sung2, Ju Ri Shin1, Young Woong Lee1, Da Jung Kim1, Kyung Seok Yang1, Sun
1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 2Biochemicals and Synthetic Biology Research Center, Korea
Research Institute of Bioscience and Biotechnology, Daejeon, Korea
Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics.
However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments.
Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with
cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without
toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via
lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known
CPP Tat (49–57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain
variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than
Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great
potential as a useful drug delivery carrier with cancer cell specificity.
Citation: Lim KJ, Sung BH, Shin JR, Lee YW, Kim DJ, et al. (2013) A Cancer Specific Cell-Penetrating Peptide, BR2, for the Efficient Delivery of an scFv into Cancer
Cells. PLoS ONE 8(6): e66084. doi:10.1371/journal.pone.0066084
Editor: Robert W. Sobol, University of Pittsburgh, United States of America
Received November 2, 2012; Accepted May 6, 2013; Published June 11, 2013
Copyright: ? 2013 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and
Technology (2011-0031955). 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.
* E-mail: email@example.com
The beneficial effects of many newly-discovered potential
therapeutic agents, such as proteins, nucleic acids, and hydrophilic
drugs, are limited because of their inability to reach the
appropriate intracellular targets [1,2]. Thus, numerous approach-
es such as microinjection, eletroporation, liposomal formulation
and the use of viral vectors have been explored to promote
efficient drug delivery [3,4]. One major concern about these
techniques is their poor cell specificity [4,5]. Therefore, the
development of a target-specific drug delivery system is a primary
concern for improving the therapeutic efficacy of drugs while
reducing their effective doses and side effects [6,7].
Cell-penetrating peptides (CPPs), also referred to as protein
transduction domains, have drawn special attention as an
alternative intracellular drug delivery vehicle since the discovery
of the first CPP, Tat, in 1988 . CPPs are short peptides
consisting of fewer than 30 amino acids and composed mostly of
basic, positively charged amino acids (e.g. Arg, Lys and His) that
have the capacity to translocate through the cell membrane and to
deliver a variety of cell-impermeable cargoes across the cellular
membrane , including proteins , nucleic acids , siRNA
, peptide nucleic acids (PNAs) , small molecule therapeu-
tics , quantum dots , and MRI contrast agents .
Although the exact mechanism of CPPs is unknown, recent
mechanistic studies imply that their cellular uptake results from an
initial rapid electrostatic interaction with the plasma membrane
followed by endosomal uptake [17,18].
Using CPPs for the intracellular delivery of a wide range of
macromolecules is a powerful approach because of their versatility
paired with easy functionalization of linked cargoes and the high
delivery efficiency into various cell lines, overcoming challenges
often faced with other delivery methods [19,20]. Therefore, many
studies have focused on the development of novel CPPs; the
number of available CPPs with different characteristics, such as
increased stability and efficient cargo delivery, continues to
Although the potential of CPPs as delivery agents is large, their
lack of cell specificity, cytotoxic effects and unexpected side effects
are major concerns for their development as drug delivery vehicles
. For cancer therapy, CPP cell specificity is especially
important so that side effects on normal cells are minimized
[22,23]. Therefore, there is a strong need for the development of
cancer-specific and non-toxic CPPs for effective cancer treatments.
We have previously reported that a potent antimicrobial
peptide, buforin IIb (RAGLQFPVG[RLLR]3), has strong cell-
penetrating ability and anticancer activity against various cancer
cell lines [24,25]. Even though buforin IIb showed selective
cytotoxicity against cancer cells, it also affected the viability of
normal cells at high concentrations. To develop buforin IIb as an
efficient drug delivery vehicle, its cytotoxicity against normal cells
should be minimized while maintaining its cancer cell specificity.
In this study, we designed a novel cancer-specific and non-toxic
cell-penetrating peptide, BR2, based on the cell-penetrating motif
of buforin IIb and studied the potential as an efficient drug
PLOS ONE | www.plosone.org1 June 2013 | Volume 8 | Issue 6 | e66084
delivery vehicle into cancer cells by fusing BR2 to a single-chain
variable fragment (scFv) antibody against mutated K-ras.
Materials and Methods
Human cervical cancer cell line HeLa, human colon cancer cell
line HCT116, mouse melanoma cell line B16/F10, mouse
fibroblast cell line NIH 3T3, human keratinocyte cell line HaCat
and human fibroblast cell line BJ were all obtained from American
Type Culture Collection (ATCC; Manassas, VA) and cultured in a
complete medium [Dulbecco’s modified eagle medium] (DMEM)
supplemented with 10% fetal bovine serum (FBS), 100 units/ml
penicillin, 100 mg/ml streptomycin. Cells were grown in humid-
ified conditions at 37uC with 5% CO2.
Peptide Design and Synthesis
We designed several derivatives of buforin IIb (BR3) by stepwise
elimination of the C-terminal regular a-helical motif RLLR
repeats of buforin IIb to create a cancer cell specific and non-toxic
CPP. The designed peptides consisted of different numbers of the
C-terminal regular a-helical motif RLLR and named BR1 and
BR2 (Table 1).
CPPs Tat, BR1, BR2, and BR3 were chemically synthesized
(Anygen, Kwangju, Korea) on a MilliGen 9050 peptide synthe-
sizer. The fluorescein moiety (FITC) was attached to the N-
terminus via an aminohexanoic acid spacer by treating a resin-
bound peptide (0.1 mM) with FITC (0.1 mM) and diisopropyl
ethyl amine (0.5 mM) in N, N-Dimethylformamide (DMF) for
12 h. All crude peptides were purified and analyzed by reversed-
phase high performance liquid chromatography (RP-HPLC) on a
C18 column, and the purified peptides were characterized by
electrospray ionization mass spectrometry (ESI-MS).
Confocal Laser Scanning Microscopy
To investigate the cell-penetrating ability and the intracellular
distribution of the internalized peptides, live confocal microscopy
was performed on three cancer lines (HeLa, HCT116 and B16/
F10) and three normal cell lines (HaCat, BJ and NIH 3T3).
Briefly, cells (26105) were plated on a glass coverslip placed in a 6-
well plate, grown overnight, and then incubated with FITC-
labeled peptides (5 mM for each cell line) for 30 min. The cells
were then rinsed three times with phosphate buffered saline (PBS,
pH 7.4), and mounted on microscope slides with fluorescence
mounting solution (Dako Corp, Carpinteria, CA). Colocalization
of BR2 with lysosomes was observed by using LysoTracker Red
DND-99 (Molecular Probe, Eugene, OR). To avoid the effects of
fixation artifacts, involving both methanol and paraformaldehyde,
cells were not fixed [26,27]. The distribution of FITC-labeled
peptides was analyzed using a confocal scanning laser Zeiss LSM
510 microscope (Jena, Germany) equipped with a 406 and 206
objective. Fluorophores were excited with an argon laser (488 nm)
for FITC and a HeNe laser (543 nm) for LysoTracker Red.
In vitro Cytotoxicity Assay
The cytotoxicity of peptides to mammalian cells was investi-
gated by assessing the release of lactate dehydrogenase (LDH)
from cancer and normal cells. The amount of LDH released from
damaged cells into the supernatant was measured using the
Cytotoxicity Detection Kit (Roche Applied Science, Germany)
according to the manufacturer’s instructions. In brief, cells were
plated onto 96-well microplates (16104cells per well) in complete
DMEM supplemented with 10% FBS and incubated overnight at
37uC to allow for attachment and spreading of cells. After 24 h of
incubation, cells were treated with various concentrations of
peptides (0–100 mM) and incubated for another 24 h at 37uC. The
extracellular medium from each well was transferred to a new
microplate and incubated for 10 min with 100 ml/well reaction
mixture, followed by a stop solution. Absorbance was measured at
490 nm by using an ELISA plate reader. LDH release from cells
lysed with 0.2% Triton X-100 in PBS was defined as 100%
leakage and LDH release from untreated cells as 0% leakage.
Hemolytic activity was assayed as described by Aboudy et al.
with slight modifications ; 3 ml of freshly prepared human
erythrocytes was washed with isotonic PBS, pH 7.4, until the color
of the supernatant turned clear. The washed erythrocytes were
then diluted to a final volume of 20 ml with the same buffer.
Peptide samples (10 ml), serially diluted in PBS, were added to
190 ml of the cell suspension in microcentrifuge tubes. Following
gentle mixing, the tubes were incubated at 37uC for 30 min and
then centrifuged at 4,0006g for 5 min. The supernatant (100 ml)
was removed to a new tube and the absorbance at 567 nm was
determined. The relative optical density, as compared with that of
the cell suspension treated with 0.2% Triton X-100, was defined as
percentage of hemolysis. The hemolysis percentage was calculated
using the following equation: percentage hemolysis=[(Abs567 nm
in the peptide solution – Abs567 nmin PBS)/(Abs567 nmin 0.2%
Triton X-100 – Abs567 nmin PBS)]6100.
Characterization of Peptide Uptake
To evaluate the internalization of FITC-labeled peptides, HeLa
cells were seeded onto 12-well plates at a density of 26105cells per
well and incubated for 24 h. FITC-labeled peptides, at various
concentrations ranging from 2 to 10 mM, were then incubated
with the cells for 30 min at 37uC. To compare the cellular uptake
of peptides, cancer and normal cells were treated with FITC-
labeled peptides (each, 10 mM) and incubated for 30 min at 37uC.
Following the incubation, cells were washed three times with ice-
cold PBS to remove excess extracellular complexes. Next, the cells
were treated with trypsin (1 mg/ml) for 10 min to remove any
remaining peptides bound to the cell surface. After trypsinization,
the cells were collected by centrifugation (1,0006g for 5 min),
resuspended with 500 ml ice-cold 2% FBS/PBS containing
propidium iodide (PI), and then immediately analyzed (10,000
events/sample) by fluorescence activated cell sorting (FACS).
To understand further the cell-penetrating mechanism of
peptides, the effects of temperature and metabolic inhibitors were
examined. To elucidate the temperature dependency, HeLa cells
were incubated at 4uC for 30 min prior to the addition of the
peptides. Next, cells were treated with FITC-labeled peptides
(each, 5 mM) at 4uC for 30 min. For the energy-depletion study,
HeLa cells were preincubated with sodium azide (NaN3,10 mM)
Table 1. Amino acid sequences of peptides used in this
Peptides Amino acid sequenceCharge Ref.
Tat (49–57)RKKRRQRRR (9 aa)
Buf IIb [BR3] RAGLQFPVGRLLRRLLRRLLR (21 aa)
BR2 RAGLQFPVGRLLRRLLR (17 aa)
BR1RAGLQFPVGRLLR (13 aa)
Underline indicates the model a-helical sequence.
A Cancer Specific Cell-Penetrating Peptide, BR2
PLOS ONE | www.plosone.org2 June 2013 | Volume 8 | Issue 6 | e66084
1. Martin I, Teixido M, Giralt E (2010) Building cell selectivity into CPP-mediated
strategies. Pharmaceuticals 3: 1456–1490.
2. Torchilin V (2008) Intracellular delivery of protein and peptide therapeutics.
Drug Discovery Today: Technologies 5: 95–103.
3. Pujals S, Giralt E (2008) Proline-rich, amphipathic cell-penetrating peptides.
Adv Drug Deliv Rev 60: 473–484.
4. Elmquist A, Lindgren M, Bartfai T, Langel U (2001) VE-cadherin-derived cell-
penetrating peptide, pVEC, with carrier functions. Exp Cell Res 269: 237–244.
5. Vives E, Brodin P, Lebleu B (1997) A truncated HIV-1 Tat protein basic
domain rapidly translocates through the plasma membrane and accumulates in
the cell nucleus. J Biol Chem 272: 16010–16017.
6. Manish G, Vimukta S (2011) Targeted drug delivery system: a review.
Res J ChemSci 1: 135–138.
7. Wang B, Siahaan TJ, Soltero R (2005) Drug Delivery: Principles and
8. Kamada H, Okamoto T, Kawamura M, Shibata H, Abe Y, et al. (2007)
Creation of novel cell-penetrating peptides for intracellular drug delivery using
systematic phage display technology originated from Tat transduction domain.
Biol Pharm Bull 30: 218–223.
9. Zorko M, Langel U (2005) Cell-penetrating peptides: mechanism and kinetics of
cargo delivery. Advanced Drug Delivery Reviews 57: 529–545.
10. Fawell S, Seery J, Daikh Y, Moore C, Chen LL, et al. (1994) Tat-mediated
delivery of heterologous proteins into cells. Proc Natl Acad Sci U S A 91: 664–
11. Johnson LN, Cashman SM, Kumar-Singh R (2008) Cell-penetrating peptide for
enhanced delivery of nucleic acids and drugs to ocular tissues including retina
and cornea. Mol Ther 16: 107–114.
12. Chiu YL, Ali A, Chu CY, Cao H, Rana TM (2004) Visualizing a correlation
between siRNA localization, cellular uptake, and RNAi in living cells. Chem
Biol 11: 1165–1175.
13. Turner JJ, Ivanova GD, Verbeure B, Williams D, Arzumanov AA, et al. (2005)
Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors
of HIV-1 Tat-dependent trans-activation in cells. Nucleic Acids Res 33: 6837–
14. Rothbard JB, Garlington S, Lin Q, Kirschberg T, Kreider E, et al. (2000)
Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery
and inhibition of inflammation. Nat Med 6: 1253–1257.
15. Yukawa H, Kagami Y, Watanabe M, Oishi K, Miyamoto Y, et al. (2010)
Quantum dots labeling using octa-arginine peptides for imaging of adipose
tissue-derived stem cells. Biomaterials 31: 4094–4103.
16. Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, et al. (2000) Tat peptide-
derivatized magnetic nanoparticles allow in vivo tracking and recovery of
progenitor cells. Nat Biotechnol 18: 410–414.
17. Heitz F, Morris MC, Divita G (2009) Twenty years of cell-penetrating peptides:
from molecular mechanisms to therapeutics. Br J Pharmacol 157: 195–206.
18. Trabulo S, Cardoso A, Mano M, Pedroso de Lima M (2010) Cell-penetrating
peptides - Mechanisms of cellular uptake and generation of delivery systems.
Pharmaceuticals 3: 961–993.
19. Hoyer J, Neundorf I (2012) Peptide vectors for the nonviral delivery of nucleic
acids. Acc Chem Res 45: 1048–1056.
20. Mussbach F, Franke M, Zoch A, Schaefer B, Reissmann S (2011) Transduction
of peptides and proteins into live cells by cell penetrating peptides. J Cell
Biochem 112: 3824–3833.
21. Kersemans V, Cornelissen B (2010) Targeting the tumour: Cell penetrating
peptides for molecular imaging and radiotherapy. Pharmaceuticals 3: 600–620.
22. Geisler I, Chmielewski J (2009) Cationic amphiphilic polyproline helices: side-
chain variations and cell-specific internalization. Chem Biol Drug Des 73: 39–
23. Martin I, Teixido M, Giralt E (2010) Building cell selectivity into CPP-mediated
strategies. Pharmaceuticals 3: 1456–1490.
24. Lee HS, Park CB, Kim JM, Jang SA, Park IY, et al. (2008) Mechanism of
anticancer activity of buforin IIb, a histone H2A-derived peptide. Cancer Lett
25. Cho JH, Sung BH, Kim SC (2009) Buforins: histone H2A-derived antimicrobial
peptides from toad stomach. Biochim Biophys Acta 1788: 1564–1569.
26. Lundberg M, Johansson M (2002) Positively charged DNA-binding proteins
cause apparent cell membrane translocation. Biochem Biophys Res Commun
27. Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, et al. (2003) Cell-
penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol
Chem 278: 585–590.
28. Aboudy Y, Mendelson E, Shalit I, Bessalle R, Fridkin M, et al. (1994) Activity of
two synthetic amphiphilic peptides and magainin-2 against herpes simplex virus
types 1 and 2. Int. J. Peptide Protein Res., 43: 6, 573–582.
29. Crombez L, Aldrian-Herrada G, Konate K, Nguyen QN, McMaster GK, et al.
(2009) A new potent secondary amphipathic cell-penetrating peptide for siRNA
delivery into mammalian cells, Molecular Therapy, 17, 95–103.
30. Bradford MM (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem 72: 248–254.
31. Shin SY, Lee SH, Yang ST, Park EJ, Lee DG, et al. (2001) Antibacterial,
antitumor and hemolytic activities of alpha-helical antibiotic peptide, P18 and its
analogs. J Pept Res 58: 504–514.
32. Chen Y, Mant CT, Farmer SW, Hancock RE, Vasil ML, et al. (2005) Rational
design of alpha-helical antimicrobial peptides with enhanced activities and
specificity/therapeutic index. J Biol Chem 280: 12316–12329.
33. Huang YB, He LY, Jiang HY, Chen YX (2012) Role of helicity on the
anticancer mechanism of action of cationic-helical peptides. Int J Mol Sci 13:
34. Leuschner C, Hansel W (2004) Membrane disrupting lytic peptides for cancer
treatments. Curr Pharm Des 10: 2299–2310.
35. Papo N, Shai Y (2003) New lytic peptides based on the D,L-amphipathic helix
motif preferentially kill tumor cells compared to normal cells. Biochemistry 42:
36. Cappelli G, Paladini S, D’Agata A (1999) Tumor markers in the diagnosis of
pancreatic cancer. Tumori 85: S19–21.
37. Madani F, Lindberg S, Langel U, Futaki S, Graslund A (2011) Mechanisms of
cellular uptake of cell-penetrating peptides. J Biophys 2011: 414729.
38. Doherty GJ, McMahon HT (2009) Mechanisms of endocytosis. Annu Rev
Biochem 78: 857–902.
39. Cawthorn TR, Amir E, Broom R, Freedman O, Gianfelice D, et al. (2009)
Mechanisms and pathways of bone metastasis: challenges and pitfalls of
performing molecular research on patient samples. Clin Exp Metastasis 26: 935–
40. Harvey TM, Emmanuel B (2011) Molecular mechanism and physiological
functions of clathrin-mediated endocytosis. Nature Reviews Molecular Cell
biology 12: 517–533.
41. Kirsten S, Sascha P, Tore S, Bo van D (2011) Clathrin-independent endocytosis:
mechanisms and function. Current Opinion in Cell Biology 23: 413–420.
42. Tanaka T, Rabbitts TH (2003) Intrabodies based on intracellular capture
frameworks that bind the RAS protein with high affinity and impair oncogenic
tramsformation. EMBO J 22: 1025–1035.
43. Deshpande AK, Kung HF (1987) Insulin induction of Xenopus laevis oocyte
maturation is inhibited by monoclonal antibody against p21 ras proteins. Mol
Cell Biol 7: 1285–1288.
44. Fox PL, Sa G, Dobrowolski SF, Stacey DW (1994) The regulation of endothelial
cell motility by p21 ras. Oncogene 9: 3519–3526.
45. Cochet O, Kenigsberg M, Delumeau I, Virone-Oddos A, Multon MC, et al.
(1998) Intracellular expression of an antibody fragment-neutralizing p21 ras
promotes tumor regression. Cancer Res 58: 1170–1176.
46. Jeyapaul J, Reddy MR, Khan SA (1990) Activity of synthetic tat peptides in
human immunodeficiency virus type 1 long terminal repeat-promoted tran-
scription in a cell-free system. Proc Natl Acad Sci U S A 87: 7030–7034.
47. Park CB, Yi KS, Matsuzaki K, Kim MS, Kim SC (2000) Structure-activity
analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline
hinge is responsible for the cell-penetrating ability of buforin II. Proc Natl Acad
Sci U S A 97: 8245–8250.
A Cancer Specific Cell-Penetrating Peptide, BR2
PLOS ONE | www.plosone.org11 June 2013 | Volume 8 | Issue 6 | e66084