Novel peptide-dendrimer conjugates as drug carriers for targeting nonsmall cell lung cancer.

Abstract

Phage display technology has been demonstrated to be a powerful tool for screening useful ligands that are capable of specifically binding to biomarkers on the surface of tumor cells. The ligands found by this technique, such as peptides, have been successfully applied in the fields of early cancer diagnostics and chemotherapy. In this study, a novel nonsmall cell lung cancer-targeting peptide (LCTP, sequence RCPLSHSLICY) was screened in vivo using a Ph.D.-C7C(™) phage display library. In order to develop a universal tumor-targeting drug carrier, the LCTP and fluorescence-labeled molecule (FITC) were conjugated to an acetylated polyamidoamine (PAMAM) dendrimer of generation 4 (G4) to form a PAMAM-Ac-FITC-LCTP conjugate. The performance of the conjugate was first tested in vitro. In vitro results of cell experiments analyzed by flow cytometry and inverted fluorescence microscopy indicated that PAMAM-Ac-FITC-LCTP was enriched more in NCI-H460 cells than in 293T cells, and cellular uptake was both time- and dose-dependent. The tissue distribution of the conjugate in athymic mice with lung cancer xenografts was also investigated to test the targeting efficiency of PAMAM-Ac-FITC-LCTP in vivo. The results showed that LCTP can effectively facilitate the targeting of PAMAM-Ac-FITC-LCTP to nonsmall cell lung cancer cells and tumors. These results suggest that the LCTP-conjugated PAMAM dendrimer might be a promising drug carrier for targeted cancer diagnosis and treatment.

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Open Access Full Text Article
Novel peptide–dendrimer conjugates as drug
carriers for targeting nonsmall cell lung cancer
DOI: 10.2147/IJN.S14601
Jianfeng Liu1,2
Jinjian Liu2
Liping chu2
Yanming Wang3
Yajun Duan1
Lina Feng2
cuihong Yang1
Ling Wang3
Deling Kong1
1The Key Laboratory of Bioactive
Materials, Ministry of education,
college of Life science, Nankai
University, Tianjian, People’s republic
of china; 2Tianjin Key Laboratory
of Molecular Nuclear Medicine,
Institute of radiation Medicine,
chinese Academy of Medical science
and Peking Union Medical college,
Tianjian, People’s republic of china;
3college of Pharmacy, Nankai
University, Tianjin, People’s republic
of china
correspondence: Deling Kong
Institute of Molecular Biology,
Nankai University, Tianjin 300071,
People’s republic of china
Tel +86 222 350 2111
Fax +86 222 349 8775
email kongdeling@nankai.edu.cn
Jianfeng Liu
Institute of radiation Medicine, Tianjin
300192, People’s republic of china
Tel +86 228 568 3019
email lewis78@163.com
Abstract: Phage display technology has been demonstrated to be a powerful tool for screening
useful ligands that are capable of specifically binding to biomarkers on the surface of tumor
cells. The ligands found by this technique, such as peptides, have been successfully applied
in the fields of early cancer diagnostics and chemotherapy. In this study, a novel nonsmall cell
lung cancer-targeting peptide (LCTP, sequence RCPLSHSLICY) was screened in vivo using
a Ph.D.-C7CTM phage display library. In order to develop a universal tumor-targeting drug
carrier, the LCTP and fluorescence-labeled molecule (FITC) were conjugated to an acetylated
polyamidoamine (PAMAM) dendrimer of generation 4 (G4) to form a PAMAM–Ac–FITC–
LCTP conjugate. The performance of the conjugate was first tested in vitro. In vitro results of
cell experiments analyzed by flow cytometry and inverted fluorescence microscopy indicated
that PAMAM–Ac–FITC–LCTP was enriched more in NCI-H460 cells than in 293T cells, and
cellular uptake was both time- and dose-dependent. The tissue distribution of the conjugate in
athymic mice with lung cancer xenografts was also investigated to test the targeting efficiency
of PAMAM–Ac–FITC–LCTP in vivo. The results showed that LCTP can effectively facilitate
the targeting of PAMAM–Ac–FITC–LCTP to nonsmall cell lung cancer cells and tumors. These
results suggest that the LCTP-conjugated PAMAM dendrimer might be a promising drug carrier
for targeted cancer diagnosis and treatment.
Keywords: polyamidoamine dendrimer, in vivo phage display, targeted drug delivery, peptide,
nonsmall cell lung cancer
Introduction
Nonspecific anticancer chemotherapy has no tumor targeting effect and will kill normal
and cancer cells, causing severe side effects in many patients. To solve this problem,
targeted drug delivery systems have received great attention.1 In this system, drugs
can be specifically targeted to the tumors, increase drug solubility,2,3 prolong time of
drugs in the circulation, protect drugs from degradation, and improve their metabolic
kinetics.4,5 To date, liposomes,6 micelles,7,8 and dendrimers9 conjugated with specific
ligands have been identified as being able to target drugs to cancer cells.
Polyamidoamine (PAMAM) dendrimers have attracted considerable research inter-
est as drug carriers due to their unique properties, ie, hyperbranched and monodispersed
tree-like structures with multifunctional surfaces, enabling the dendrimers to encapsu-
late or conjugate drug molecules. Moreover, the terminal groups of PAMAM can be
easily modified (by, eg, acetylation or poly(ethyleneglycol) modification [PEGylation])
to improve their solubility and biocompatibility. In order to improve the targeting capa-
bility of PAMAM, many targeting molecules, such as antibodies,10 folic acid,11,12 biotin,13
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Liu et al
peptides,14–17 and carbohydrates18,19 have been connected to
PAMAM dendrimers. Finally, chemotherapy drugs have been
covalently bound to these dendrimers20,21 or encapsulated in
the internal cavities of dendritic molecules11,22 to generate
targeted drug delivery systems.
A key factor of a targeted drug delivery system is the
finding of targeting molecules that can specifically recognize
tumor and cancer cells. Phage display technology is a powerful
approach to screening targeting molecules, such as peptides,
for cancer cells or tumor blood vessel endothelial cells.23,24
Many novel angiogenic vessels and homing peptides have
been isolated recently using this method.17,25,26 Furthermore,
in vivo phage display can screen cancer-binding peptides
regardless of whether the receptor is known or not. Therefore,
this technique can quickly screen cancer-specific peptides.27,28
In addition to drug delivery systems, tumor-targeting peptides
are also employed in diagnosis or radiotherapy by delivering
radionuclide.29–31 Some peptides discovered by phage display
are even able to inhibit cancer growth and induce cancer cell
apoptosis.32,33
In order to develop a universal drug carrier for nonsmall
cell lung cancer chemotherapy, a Ph.D.-C7CTM (New England
Biolabs, Beverly, MA) phage display library was utilized to
screen peptides specific for nonsmall cell lung cancer. It has
become generally accepted that organs have their own special
markers. Such heterogeneity was reported after it became
apparent that endothelial cells in different organs display
organ-specific markers. The phage display library includes
all possible peptide sequences, and some random peptides
displayed by phage can bind specifically with tumor-specific
markers displayed by nonsmall cell lung cancer, based on
protein–protein interactions. The phage displaying specific
peptides would home to the tumor site, after intravenous
injection. The tumor is then harvested and the phage therein
collected and amplified. After four to five rounds, the phage
including the tumor-specific targeting peptide can be isolated
and the inserted peptide sequence translated by the phage
DNA sequence. In our study, a novel peptide, lung cancer-
targeting peptide (LCTP; RCPLSHSLICY), was discovered
which can specifically target nonsmall cell lung cancer. The
LCTP was then conjugated with fluorescein isothiocyanate
(FITC, a fluorescence labeling agent used as a tracer) and
acetylated PAMAM to generate a targeted drug delivery
carrier (PAMAM–Ac–FITC–LCTP). The performance of
this drug carrier was evaluated by in vitro culturing NCI-
H460 and 293T cells and in vivo using athymic mice with
lung cancer xenografts. Our results showed that the modified
PAMAM peptide dendrimer could be easily taken up by
NCI-H460 cells in vitro and by tumors in vivo.
Materials and methods
Materials
Phenyl methyl sulphonyl fiuoride (PMSF), isopropyl β-D-1-
thiogalactopyranoside (IPTG), aprotinin, leupeptin, 5-bromo-
4-chloro-3- indolyl-β-D-galactopyranoside (X-gal), FITC,
5,5-dithiobis-2-nitrobenzoic acid (DTNB), and polyamido-
amine (G 4) were purchased from Sigma-Aldrich (St Louis,
MO). Dialysis membrane (MWCO, 3500) was purchased
from BBI Inc (Shanghai, China). 3-(4,5-dimethylthiazol-2-
yl)-2,5- diphenyl tetrazolium bromide (MTT), all cell culture
media, and supplies were purchased from Gibco Corporation
(Grand Island, NE).
The NCI-H460 cell line was kindly provided by Dr Yong
Wang (Institute of Radiation Medicine, Chinese Academy of
Medical Sciences, Tianjin, China). The 293T cell line was
purchased from Keygen Co (Nanjing, China). Phage DNA
sequencing was performed by Shanghai Sangon Corp (Shang-
hai, China). Peptides were synthesized by GL Biochem Ltd
(Shanghai, China). BALB/c-nu/nu athymic mice (female, 4–6
weeks old) were purchased from the Laboratory Animal Cen-
ter of The Academy of Military Medical Sciences (Beijing,
China). The animal studies were performed in accordance
with the Regulations for the Administration of Affairs Con-
cerning Experimental Animals (Tianjin, revised in June 2004)
and adhered to the Guiding Principles in the Care and Use of
Animals of the American Physiological Society. The Ph.D.-
C7CTM phage display peptide library kit was used to screen
specific peptides binding to human lung cancer xenografts
on BALB/c-nu/nu athymic mice.
The phage display library kit is based on a combinatorial
library of random peptide 7-mers fused to the N terminus of
the minor coat protein (cpIII) of M13 phage. The random
sequence is flanked by a pair of cysteine residues. Under
nonreducing conditions, the cysteines spontaneously form
a disulfide crosslink, resulting in phage display of cyclized
peptides. The titer of the library is 2 × 1013 plaque-forming
units (pfu). The library consists of 1.2 × 109 electroporated
sequences (compared with 207 = 1.28 × 109 possible seven-
residue sequences). Extensively sequencing the naive library
has revealed a wide diversity of sequences with no obvious
positional biases. The Escherichia coli host strain ER2738
(Arobust F+ strain with a rapid growth rate; New England
Biolabs) was used for M13 phage propagation.
In vivo phage display screening
and peptide synthesis
BALB/c-nu/nu athymic mice (female, 4–6 weeks) were
used, and 2 × 107 NCI-H460 cells in 0.2 mL of Roswell
Park Memorial Institute 1640 (RPMI-1640) medium were

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Peptide–dendrimer drug carriers for NscLc
subcutaneously injected in the right flank to produce lung
cancer xenografts. A Ph.D.-C7C phage display peptide
library, which contained 2 × 1011 pfu was mixed with
200 µL of Dulbecco’s modified Eagle’s medium (DMEM),
and injected into the mice through the tail vein when the
tumor grew up to about 0.5 cm in diameter. After circula-
tion for 5 minutes, the mice were sacrificed, and perfused
by injection of 50 mL phosphate-buffered saline through
the heart to wash the unbounded phage. Then the tumors
were harvested, weighed, and ground in 1 mL of DMEM-PI
(DMEM containing the protease inhibitors PMSF [1 mM],
aprotinin [20 µg/mL], and leupeptin [1 µg/mL]). The
tissue was washed five times with ice-cold DMEM-PI
containing 1% bovine serum albumin. After centrifugation,
the phage particles were amplified by ER2738 bacteria
overnight at 37°C. The phages were titered on agar plates
in the presence of IPTG/X-gel (1 mg/L) and tetracycline
(40 µg/mL). The amplified phage was injected into the
mice to repeat the above procedures. This screening was
repeated for five rounds.17,27,34 At the end of the fifth round,
the phage was eluted and titered on LB/ IPTG/X-gel
plates. The phage clones were randomly selected, and
the inserted DNA sequence was determined using primer
5′-CCCTCATAGTTAGCGTAACG-3′ (New England Bio-
labs). The phage-displayed peptides were translated and
synthesized according the DNA sequences.
synthesis of acetylated PAMAM
dendrimer
The acetylated PAMAM dendrimer (PAMAM–Ac) was
synthesized according to the method described in the
literature.13,16,35 Briefly, 1.012 g (MW 14215, 71.2 µmol)
of PAMAM dendrimer in 120 mL anhydrous methanol
(MeOH) was reacted with 0.214 g (2.110 mmol) of acetic
anhydride overnight at room temperature with stirring in the
presence of 0.352 g (3.454 mmol) of triethylamine. After
evaporation, the product was dialyzed for 3 days against
phosphate-buffered saline and double-distilled water using
the dialysis membrane (MWCO = 3500 Da), then lyo-
philized, and 1.165 g of PAMAM–Ac was obtained. The
average number of acetyl groups grafted to each PAMAM
molecule was determined by 1H nuclear magnetic resonance
calibration.
conjugation of FITc to acetylated
PAMAM dendrimer
FITC (38.2 mg, 98.1 µmol) was added dropwise into 40 mL of
dimethyl sulfoxide (DMSO) solution containing 0.401 g (MW
15433, 25.9 µmol) of PAMAM–Ac. The reaction mixture was
allowed to stir overnight. The product was dialyzed against
phosphate-buffered saline and double-distilled water using
dialysis membrane (MWCO = 3500 Da) for 3 days in a
dark room, and the adsorbance at 500 nm of the eluent was
detected to ensure that all free FITC was removed. The
product was then lyophilized in a dark room, and 0.387 g
of PAMAM–Ac–FITC was obtained. The a verage number
of FITC molecules conjugated to each PAMAM molecule
was determined by 1H nuclear magnetic resonance calibra-
tion and ultraviolet-visible adsorbance (Varian Cary 100
bio ultraviolet-visible spectrophotometer).
conjugation of peptides to FITc-labeled
dendrimer
The solution containing 9.1 mg (90.9 µmol) of succinic
anhydride in 6 mL of anhydrous MeOH was added drop-
wise into a solution of 0.103 g (MW 16601, 6.2 µmol) of
PAMAM–Ac–FITC and 24.5 mg of (0.242 mmol) trietha-
nolamine (TEA) in 90 mL of anhydrous MeOH with stirring.
After overnight reaction at room temperature, the solvent
was evaporated, and then the crude product was dialyzed
(MWCO = 3500 Da) and lyophilized using the aforemen-
tioned method; 12.8 mg (MW 18001, 0.71 µmol) of the
above product was then reacted with 2.0 mg (10.4 µmol)
of 1-[3-(dimethylamino)-propyl]-3-ethyl-carbodimide HCl
(EDC) in 4 mL of water for 3 hours. Then 9.2 mg (7.1 µmol)
of the LCTP in 1 mL of DMSO was added dropwise to the
above solution and stirred overnight. The crude product was
dialyzed (MWCO = 3500 Da) using the aforementioned
method, and the adsorbance at 275 nm of the elute was
detected to ensure that all free peptide was removed. Then the
product was lyophilized in a dark room. The average number
of peptides conjugated to the dendrimer was determined
using the Ellman assay.36 A standard curve was plotted with
free LCTP. During the process, an excess of sodium sulfite
was added to the peptide solution at pH 9.0 to cleave the
disulfide bond, and the reaction was carried out in a dark
room. The adsorbance at 412 nm was used to calculate the
number of peptides conjugated to the dendrimer according
to the standard curve.
Laser light-scattering assay
Samples of PAMAM–Ac, PAMAM–Ac–FITC, and PAMAM–
Ac–FITC–LCTP were filtered (pore size, 0.45 mm) and
analyzed with a light-scattering spectrometer (BI-200SM;
Brookhaven Instruments, Holtsville, NY), equipped with a
digital correlator (BI-9000AT; Brookhaven Instruments) to
determine the granulometric distribution of the conjugates
at 532 nm.

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Liu et al
cell culture
NCI-H460 cells were cultured in RPMI-1640 medium and
293T cells in DMEM medium. The medium was supplemented
with 10% fetal bovine serum, 1% penicillin, and 100 µg/mL
streptomycin. Cells were grown at 37°C in 5% CO2.
cytotoxicity assay
The cytotoxicity of the PAMAM, PAMAM–Ac, PAMAM–
Ac–FITC, and PAMAM–Ac–FITC–LCTP were evaluated
by the MTT assay. The 293T cells were seeded into 96-well
plates at a density of 104 cells/well and incubated for 24 hours.
The filtrate of the samples (0.22 µm) was added to the cells
at different concentrations (0, 0.125, 0.25, 0.5, 1.0, 2.0, and
4.0 µM). After incubation for another 24 hours, 25 µL of
MTT solution (5 mg/mL in phosphate-buffered saline) was
added to each well. Four hours later, the medium containing
MTT was removed, and the samples in the wells were air
dried. Acidic isopropanol (100 µL, 0.04 M HCl in absolute
isopropanol) was added to dissolve the formazen crystals. The
optical density of the solution was measured at 570 nm using a
microplate reader (Labsystem; Multiskan, Ascent, Finland).
In vitro cellular uptake assay
293T and NCI-H460 cells were seeded in 24-well plates at
a density of 104 cells/well. After 24 hours, the cells were
incubated with PAMAM–Ac–FITC or PAMAM–Ac–FITC–
LCTP at different concentrations ranging from 0.05 to
1.0 µM for 4 hours, or incubated with 0.25 µM of PAMAM–
Ac–FITC–LCTP at different intervals ranging from 0.5 to
8.0 hours. To compare the uptake efficiency between 293T
cells and NCI-H460 cells, the two cell lines were incubated
with 0.5 µM of the conjugates for 4 hours. To examine the
competitive uptake, 293T cells and NCI-H460 cells were
preincubated with 20 µM free LCTP for 1 hour before the
conjugates were added. At the end of incubation, the cells
were trypsinized, washed with physiologic saline three times,
and we then proceeded to fluorescence-activated cell sorting
(FACS) analysis (Altra FCM; Beckman Coulter, Miami, FL).
The uptake efficiency was expressed as the percentage of
FITC-positive cells. The cells were also viewed under a con-
focal microscope (DMI6000B; Leica, Wetzlar, Germany) and
an inverted fluorescence microscope. Images were recorded
using CCD (ECLIPSE TE2000-U; Nikon, Tokyo, Japan).
In vivo targeting assay
BALB/c-nu/nu athymic mice with lung cancer xenografts were
prepared as described earlier. Mice were used for experiment
when the diameter of tumor xenografts reached 0.4–0.6 cm.
The animals were randomly divided into two groups. A 0.3 mL
solution of PAMAM–Ac–FITC or PAMAM–Ac–FITC–LCTP
(10 µM in phosphate-buffered saline) was injected through
the tail vein of the animal. Four hours later, the tumors and
main organs (heart, liver, lung, spleen, kidney, thyroid,
brain, and ovary) were harvested and proceeded to imaging
(KODAK IS in vivo FX; Kodak, New Haven, CT). The rela-
tive fluorescence intensity per unit area was calculated using
KODAK 5.1 software (KODAK IS in vivo FX).
statistical analysis
The unpaired Student’s t-test was used to evaluate the sig-
nificance between experimental groups. A value of P , 0.05
was considered to be statistically significant.
Results
screening and peptide synthesis
After five rounds of screening, 40 random phage clones
were amplified and the DNA sequences were determined.
The phage displayed peptide sequences were translated
(see Table 1). The most frequent peptide sequence was
ACPLSHSLIC, and the binding sequence with nonsmall
cell lung cancer was the sequence between the two cysteine
residues. In order to connect the peptide to the PAMAM
dendrimer, the alanine residue was replaced by arginine.
For 125I-labeling, a tyrosine residue was added to the end
of the peptide. The final peptide sequence synthesized was
RCPLSHSLICY, which was cyclized with two cysteine
residues, marked LCTP (see supporting information for the
HPLC and MS spectra of the synthesized peptide).
Preparation of PAMAM–Ac–FITc–LcTP
conjugate
The synthetic route of PAMAM–Ac–FITC–LCTP con-
jugate is shown in Figure 1. The degree of PAMAM–Ac
Table 1 The displayed peptide sequences of screening phage
from in vivo lung cancer xenografts
Phage clonePeptide sequence Frequency
A-2
A-7
B-11
A-6
A-13
B-3
B-7
AcPLshsLIc
AcsVAPDNLc
AcAQsYhVWc
AcWFsNIAKc
AcshFVYgIc
AcVNgrMTDc
AcPLKANLsc
28/37
3/37
2/37
1/37
1/37
1/37
1/37
Notes: Forty phage clones were random selected from two plates (A and B) and
37 phage displayed peptide sequences were identified. The first, second, and tenth
residues are alanine, cysteine, and cysteine, respectively. The inner random seven
peptides were cyclized by the two cysteine residues.

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Peptide–dendrimer drug carriers for NscLc
acetylation was measured by 1H nuclear magnetic resonance
(300 mHz, D2O); the aliphatic peaks appear at 1.80 ppm,
2.24 ppm, 2.46 ppm, 2.64 ppm, 2.93 ppm, 3.12 ppm,
3.23 ppm, and 3.30 ppm. The integration ratio of methyl-
ene protons signal of –CH2C(O)– at δ2.24 ppm (248 H) and
methyl protons signal of –C(O)CH3 at δ1.80 ppm was 2.84,
indicating that about 29 acetyl groups were grafted to each
PAMAM molecule (PAMAM–Ac29). Conjugation of FITC
to PAMAM–Ac was characterized by 1H nuclear mag-
netic resonance (300 mHz, D2O) and ultraviolet–visible
spectrum. The aliphatic peaks appear at 1.80 ppm,
2.23 ppm, 2.44 ppm, 2.63 ppm, 2.94 ppm, 3.11 ppm,
3.29 ppm, and 3.30 ppm. The aromatic peaks at 6.37 ppm,
6.96 ppm, 7.42 ppm, and 7.67 ppm corresponded to
the protons in FITC. FITC has a characteristic absorbance
peak at around 500 nm and not for PAMAM–Ac and
the peptide ( Figure 2). The number of FITC conjugated
to PAMAM–Ac was about 3–4, which was determined
based on the standard calibration curve of free FITC.
The number of LCTP grafted to PAMAM–Ac–FITC was
7–8 by Ellman assay (see supporting information for the
1H nuclear magnetic resonance spectra of PAMAM–Ac,
PAMAM–Ac–FITC, and PAMAM–Ac–FITC–LCTP).
cytotoxicity assay
As shown in Figure 3, when the concentration of the polymers
was higher than 2 µM, the cytotoxicity of PAMAM–Ac,
PAMAM–Ac–FITC, and PAMAM–Ac–FITC–LCTP was
lower than that of unmodified PAMAM. The cells viability
was over 88%, which suggests that the PAMAM–Ac–FITC–
LCTP conjugate has good biocompatibility.
In vitro cellular uptake assay
Figure 4A shows the uptake efficiencies of the two conju-
gates for 293T cells over 4 hours. The uptake efficiencies
of both conjugates were nearly of equal value and were
200
0.0
0.1
0.2
0.3
0.4
0.5
0.6
300400500600
Wavelengths (nm)
Absorbance
1-- FITC
2-- LCTP
3-- PAMAM–Ac
4-- PAMAM–Ac–FITC
5-- PAMAM–Ac–FITC–LCTP
1
2
4
5
Figure 2 Ultraviolet-visible spectra of the synthesized polyamidoamine conjugates.
The peak at 500 nm indicates the presence of fluorescence-labeling in the conjugates.
The peptide adsorbance peak at 275 nm was overlapped with the broad adsorbance
peak of fluorescence-labeling at 275 nm.
(NH2)64
(NH2)35
(NH2)35
(FITC)3
(FITC)3
O
(FITC)3
(FITC)3
(FITC)3
FITC
DMSO
(NH2)32
(NH2)32
(NH2)18
O
(NH2)18
O
(NH2)18
O
Ac2O
MeOH
O
(NH –– C ––CH3)29
O
(NH –– C ––CH3)29
O
(NH –– C ––CH3)29
(NH –– C ––CH3)29
(NH –– C ––CH3)29
(NH –– C ––CH3)29
(NH –– C ––CH3)29
(Succinic acid)14
(Succinic acid)14
Succinic
anhydride
Peptide
(Peptide)7
Figure 1 Synthetic scheme of polyamidoamine dendrimer generation 4 fluorescence-
labeled acetylated polyamidoamine lung cancer targeting peptide.
0
0
20
40
60
80
100
120
140
1234
Concentration (µM)
Viability (%)
PAMAM
PAMAM–Ac
PAMAM–Ac–FITC
PAMAM–Ac–FITC–LCTP
Figure 3 MTT assay for cellular toxicity of 293T cells. cells were incubated with
polyamidoamine, fluorescence-labeled acetylated
polyamidoamine and fluorescence-labeled acetylated polyamidoamine lung cancer
targeting peptide, at varied concentrations. The viability of the nontreated cells was
arbitrarily defined as 100%. Data are expressed as mean ± standard deviation (n = 6).
polyamidoamine, acetylated