Oligopeptide delivery carrier for osteoclast precursors

Article abstract of DOI:10.1021/bc100066k

Dendritic amine and guanidinium group-modified nanoparticles were investigated for the delivery of model peptide drug into primary osteoclast precursor cells (bone marrow macrophages; BMMs). The model peptide drug was encapsulated into the nanoparticle by

Full text of DOI:10.1021/bc100066k

Bioconjugate Chem. 2010, 21, 1473–1478
1473
Oligopeptide Delivery Carrier for Osteoclast Precursors
Bo Chi,† So Jeong Park,‡ Min Hee Park,† Soo Young Lee,*,‡ and Byeongmoon Jeong*,†
Department of Chemistry and Nano Science and Division of Life and Pharmaceutical Sciences, Department of Bioinspired
Science, Ewha Womans University, Seoul, Korea. Received February 2, 2010; Revised Manuscript Received June 2, 2010
Dendritic amine and guanidinium group-modified nanoparticles were investigated for the delivery of model peptide
drug into primary osteoclast precursor cells (bone marrow macrophages; BMMs). The model peptide drug was
encapsulated into the nanoparticle by dropping the drug/carrier dissolved in dimethylsulfoxide/methylene chloride
cosolvent into water containing poly(vinyl alcohol) as a stabilizer. Flow cytometry and spectrofluorimetry analysis
indicated that the model drug itself was not taken up by the BMMs; however, nanoparticle systems underwent
significant cellular uptake. In particular, guanidinium group-modified nanoparticles were taken up more efficiently
than amine group-modified ones. Cell viability studies showed that both amine and guanidinium group-modified
nanoparticles exhibited no significant cytotoxicity up to 100 µg/mL against the cells.
stearyl group is used to encapsulate the hydrophobic model
oligopeptide drug, an FITC conjugated-Ile-Ile-Leu-Ala-Val-Tyr
(FITC-IILAVY). Internalization of the model oligopeptide drug
into the BMMs was investigated using a series of these
nanoparticles carriers.
INTRODUCTION
Cellular communications between osteoblasts and osteoclasts
are the basic mechanism of bone homeostasis. Increased activity
of osteoclasts relative to osteoblasts leads to diseases such as
osteoporosis, periodontitis, and arthritis (1, 2). The inhibition
of differentiation from precursor cells (bone marrow macroph-
ages; BMMs) into osteoclasts was suggested to be a promising
therapeutic treatment for such diseases (3). We have been
developing oligopeptides that inhibit osteoclast differentiation
(4). However, they need a carrier system to deliver the drug
into the BMMs; otherwise, it cannot enter the cells due to the
large size and charge of the drug.
Plasma membrane serves as the protective barrier against
extraneous molecules into living cells. To overcome the cell
membrane barriers, cell penetrating peptides (CPPs) have been
extensively investigated for the plasmid DNA, oligonucleotide,
si-RNA, peptide-nucleic acid (PNA), proteins, liposomes, and
iron nanoparticles (5, 6). TAT, penetratin, model amphipathic
peptide (MAP), transportan, vascular endothelial cadherin
derived CPP (pVEC), pISL derived from the homeodomain of
the rat transcription factor Islet-1, oligoarginine, KETWWETW-
WTEWSQPKKKRKV (Pep-1), and MANLGYWLLALFVT-
MWTDVGLCKKRPKP (PrPc) are typical examples of the
CPPs. The structural characteristics of these CPPs are positively
charged amino acids such as lysine or arginine in the peptide
sequence. In particular, guanidinium moieties are intensively
studied by Futaka and Wender, focusing on the dependence of
molecular weight of oligoarginine, topological variation into
linear and dendritic oligoarginine (7-11). The optimal number
of Arg with 8-9 is critical, because it determines the toxicity,
translocation ability of the conjugated drug, and cost of the
system (9, 10).
EXPERIMENTAL PROCEDURES
Materials. FITC-IILAVY was obtained from Peptron (Ko-
rea). N-Carboxyanhydride of DL-phenylalanine (DL-Phe-NCA)
was purchased from M&H laboratories (Korea). R,ε-Di-tert-
butyloxycarbonyl-L-lysine (R,ε-diboc-Lys) was purchased from
Shanghai GL Biochem Ltd. (China). Stearylamine and 1-ethyl-
3-(3-dimethyl aminopropyl) carbodiimide (EDC) were pur-
chased from TCI (Japan). N-Hydroxysuccinimide (NHS), 4-guani-
dinobutyricacid(GBA),dimethylsulfoxide(DMSO),paraformaldehyde,
and anhydrous N,N-dimethyl formamide (DMF) were purchased
from Sigma (America). Trifluoroacetic acid (TFA) and 4-mor-
pholine ethane sulfonic acid (MES) were purchased from Sigma
(America) and used as received.
Synthesis of SF-A1. Stearylamine in anhydrous DMF (5.3
mL, 1.0 M) was added to a DL-Phe-NCA 3.06 g (16.0 mmol)
solution dissolved in anhydrous DMF (20 mL), and the reaction
was stirred 4 days at 40 °C under N2 protection (12). The
mixtures were precipitated in diethyl ether, and the precipitate
was washed throughout with diethyl ether. The product was
dried in vacuo.
[M-Na+]: 733.7 (calc. 734.0). 1H NMR (CF3COOD): δ (ppm)
stearylamine (CH3-) ) 0.7-1.0; δ stearylamine (-CH2-) )
1.0-1.5; δ steraylamine (-CH2NH-) ) 3.0-3.1; DL-phenylanine
(-CH2-) ) 3.1-3.4; δ DL-phenylanine (-COCH(CH2C6H5)-)
) 4.6-5.0; δ DL-phenylanine (-C6H5) ) 6.8-7.4.
Synthesis of SF-G1. EDC (0.57 g, 3.0 mmol) and NHS (0.34
g, 3.0 mmol) were added to the GBA (0.43 g, 3.0 mmol) that
was dissolved in MES buffer (pH ) 5.5, 10 mL). The reaction
mixtures were stirred for 4 h. To couple the amino group of
the SF-A1 and carboxylic acid group of GBA, SF-A1 (1.51 g,
1.80 mmol) dissolved in DMF (30 mL) was slowly injected to
the reaction mixtures at 0 °C (13). The reaction was stirred at
room temperature for 48 h. The reaction mixtures were
precipitated into an excess amount of diethyl ether. The
precipitate was filtered and washed with diethyl ether three
times. After being dried in vacuo, the product was suspended
in 20 mL of deionized water and dialyzed using cellulose
In this research, we designed a series of dendritic amine and
guanidinium modified molecules, where a hydrophobic stearyl
group is connected to the amine or guanidinium groups, and
oligophenylalanine is used as a connecting moiety to give
structural rigidity to the system (Figure 1). The hydrophobic
* To whom correspondence should be addressed. B. Jong: Fax
82232772384; Tel 82232773411; E-mail bjeong@ewha.ac.kr. S. Lee:
Fax 82232773760; Tel 82232773770; E-mail leesy@ewha.ac.kr.
† Department of Chemistry and Nano Science.
‡ Division of Life and Pharmaceutical Sciences.
10.1021/bc100066k  2010 American Chemical Society
Published on Web 07/19/2010

1474 Bioconjugate Chem., Vol. 21, No. 8, 2010
Chi et al.
Figure 1. Synthetic scheme of carriers. R is stearyl (C17) group. n ≈ 3.
membrane (MWCO 500 Da) in deionized water for 24 h. The
final product was lyophilized in a powder form.
amount of diethyl ether to precipitate the product. The precipitate
was filtered and washed with diethyl ether three times. After
the residual solvent was removed in vacuo, the product was
redissolved in 20 mL deionized water and dialyzed using
cellulose membrane (MWCO 500 Da) in deionized water for
24 h. The final product was collected by lyophilization.
MALDI-TOF Mass spectra [M-H+]: 1094.0 (calc. 1093.5).
1H NMR (CF3COOD): δ (ppm) stearylamine (CH3-) ) 0.7-0.9;
δ stearylamine (-CH2-) ) 1.0-1.5; δ lysine (-COCH-
(CH2CH2CH2CH2(NH)-) ) 1.6-1.7; δ lysine (-COCH-
(CH2CH2CH2CH2(NH)-), and GBA (-OCCH2CH2CH2NH-) )
1.8-2.0; δ stearylamine (-CH2NH-) ) 3.0-3.1; δ lysine
(-COCH(CH2CH2CH2CH2(NH)-) ) 3.0-3.2; δ DL-phenyla-
nine (-CH2-) ) 3.1-3.4; δ GBA (-CH2NHC(dNH)NH-) )
3.3-3.4; δ lysine (-COCH(CH2CH2CH2CH2NH2)(NH-)), and
DL-phenylanine (-COCH(CH2C6H5)-) ) 4.7-5.0; δ DL-phe-
nylanine (-C6H5) ) 7.0-7.4.
Synthesis of SF-A4. R,ε-Diboc-Lys (4.83 g, 14.0 mmol) was
dissolved in MES buffer (30 mL, pH ) 5.5). EDC (2.70 g,
14.1 mmol) and NHS (1.65 g, 14.0 mmol) were added to the
solution, and the mixtures were stirred for 4 h. SF-A2 (3.15 g,
3.8 mmol) dissolved in DMF (30 mL) was injected to the
mixture at 0 °C, and the reaction mixtures were stirred at room
temperature for 48 h. An excess amount of diethyl ether was
added to precipitate the SF-A2-R,ε-diboc-Lys. The precipitate
was filtered and washed with diethyl ether three times. After
drying the product in vacuo, SF-A2-R,ε-diboc-Lys (5.14 g, 3.4
mmol) was stirred in a mixed solution of TFA (10 mL) and
anhydrous dichloromethane (20 mL) at 0 °C for 3 h. After the
solvent was partially evaporated under reduced pressure, an
excess amount of diethyl ether was added to precipitate the
product. The precipitate was filtered and washed with diethyl
ether three times. After the residual solvent was removed in
vacuo, the product was redissolved in 20 mL deionized water
and dialyzed using a cellulose membrane (MWCO 500 Da) in
deionized water for 24 h. The final product was collected by
lyophilization.
MALDI-TOF Mass spectra [M-H+]: 839.0 (calc. 839.2). 1H
NMR (CF3COOD): δ (ppm) searylamine (CH3-) ) 0.7-1.0; δ
stearylamine (-CH2-) ) 1.0-1.5; δ GBA (-OCCH2CH2CH2NH-)
) 1.8-2.0; δ stearylamine (-CH2NH-) ) 3.0-3.1; δ DL-
phenylanine (-CH2-) ) 3.1-3.4; δ GBA (-CH2NHC(dNH)NH2)
) 3.3-3.4; δ DL-phenylanine (-COCH(CH2C6H5)-) ) 4.6-5.0;
δ DL-phenylanine (-C6H5) ) 6.8-7.4.
Synthesis of SF-A2. R,ε-Diboc-Lys (2.41 g, 7.0 mmol) was
dissolved in MES buffer (pH ) 5.5, 10 mL), and then EDC
(1.34 g, 7.0 mmol) and NHS (0.81 g, 7.0 mmol) were added to
the solution, and the mixture was stirred for 4 h. A solution of
SF-A1 (4.00 g, 4.8 mmol) in DMF 30 mL was injected into the
mixture at 0 °C. The reaction was stirred at room temperature
for 48 h. The protective t-butoxycarbonyl groups were removed
by TFA. The SF-A1-diboc-lysine (4.34 g, 4.2 mmol) was stirred
in a mixed solution of TFA (10 mL) and anhydrous dichlo-
romethane (20 mL) at 0 °C for 3 h. After the solvent was
partially evaporated under reduced pressure, an excess amount
of diethyl ether was added to precipitate SF-A2. The precipitate
was filtered and washed with diethyl ether three times. After
being dried in vacuo, the product was dissolved in 20 mL
deionized water and dialyzed using cellulose membrane (MWCO
500 Da) in deionized water for 24 h. The final product was
collected by lyophilization.
MALDI-TOF Mass spectra [M-H+]: 839.9 (calc. 839.2).
1H NMR (CF3COOD): δ (ppm) stearylamine (CH3-) )
0.7-0.9; δ stearylamine (-CH2-) ) 1.0-1.5; δ lysine
(-COCH(CH2CH2CH2CH2(NH2))
(-COCH(CH2CH2CH2CH2(NH2))
)
)
1.6-1.7;
1.8-2.0;
δ
δ
lysine
lysine
(-COCH(CH2CH2CH2CH2(NH2)) ) 2.8-3.2; δ stearylamine
(-CH2NH-) ) 3.0-3.1; δ DL-phenylanine (-CH2-) ) 3.1-3.4;
δ lysine (-COCH(CH2CH2CH2CH2NH2)(NH2)); and DL-
phenylanine (-COCH(CH2C6H5)-) ) 4.6-5.0; δ DL-pheny-
lanine (-C6H5) ) 6.8-7.4.
Synthesis of SF-G2. GBA (1.30 g, 9.0 mmol) was dissolved
in MES buffer (10 mL at pH ) 5.5). EDC (1.77 g, 9.0 mmol)
and NHS (1.0 g, 9.0 mmol) were added to the solution, and the
mixtures were stirred for 4 h at room temperature. SF-A2 (3.15
g, 3.8 mmol) in DMF (30 mL) was injected into the mixture at
0 °C, and the reaction mixtures were stirred at room temperature
for 48 h. The reaction mixtures were poured into an excess
MALDI-TOF Mass spectra [M-Na+]: 1118.0 (calc. 1118.6).
1H NMR (CF3COOD): δ (ppm) stearylamine (CH3-) ) 0.7-0.9;
δ stearylamine (-CH2-) ) 1.0-1.5; δ lysine (-COCH-
(CH2CH2CH2CH2(NH2)) ) 1.6-1.7; δ lysine (-COCH-
(CH2CH2CH2CH2(NH2)) ) 1.8-2.0; δ lysine (-COCH-
(CH2CH2CH2CH2(NH2)) ) 2.8-3.2; δ stearylamine (-CH2NH-)

Bioconjugate Chem., Vol. 21, No. 8, 2010 1475
) 3.0-3.1; δ DL-phenylanine (-CH2-) ) 3.1-3.4; δ lysine
(-COCH(CH2CH2CH2CH2NH2)(NH2)); and DL-phenylanine
(-COCH(CH2C6H5)-) ) 4.6-5.0; δ DL-phenylanine (-C6H5) )
6.8-7.4.
ng/mL recombinant human macrophage-colony stimulating
factor (rhM-CSF) (R&D system, Minneapolis, MN).
Fluorescence Image Assay. BMMs were incubated in
R-MEM containing drug-loaded nanoparticles for 4 h. The cells
were washed twice with phosphate buffered saline (PBS).
Culture dishes were transferred to a Zeiss Axiovert 200
microscope (Carl Zeiss, Oberkochen, Germany) equipped with
AXioCam HR.
Fluorescence Activated Cell Sorting (FACS). BMMs were
seeded in 6-well plates at a density of 5 × 105 cells per well
and incubated for 24 h. BMMs were grown to approximately
60% confluency in 6-well plates and then incubated with
nanoparticles for 4 h. After the cells were washed twice with
PBS and trypsinized for 3 min, the cells were harvested and
washed twice with PBS. The cells were fixed by paraformal-
dehyde aqueous solution (2.0%) and analyzed by flow cytometry
(FACS Calibur: BD Biosciences, San Jose, CA, USA). The
excitation wavelength was set at 488 nm.
Synthesis of SF-G4. GBA (2.63 g, 18 mmol) was dissolved
in MES buffer (pH ) 5.5; 10 mL). EDC (3.62 g, 18.9 mmol)
and NHS (2.10 g, 18.3 mmol) were added to the solution, and
the mixtures were stirred for 4 h. SF-A4 (4.00 g, 3.7 mmol)
dissolved in DMF (30 mL) was injected to the mixture at 0 °C,
and the reaction mixtures were stirred at room temperature for
48 h. The reaction mixtures were precipitated into an excess
amount of diethyl ether. The precipitate was filtered and washed
with diethyl ether three times. After the residual solvent was
removed in vacuo, the product was redissolved in 20 mL
deionized water and dialyzed using cellulose membrane (MWCO
1000 Da) in deionized water for 24 h. The final product was
collected by lyophilization.
MALDI-TOF Mass spectra [M-H+]: 1608.6 (calc. 1608.1).
1H NMR (CF3COOD): δ (ppm) stearylamine (CH3-) ) 0.7-0.1.0;
δ stearylamine (-CH2-) ) 1.0-1.5; δ lysine (-COCH-
(CH2CH2CH2CH2(NH)-) ) 1.6-1.7; δ lysine (-COCH-
(CH2CH2CH2CH2(NH)-), and GBA (-OCCH2CH2CH2NH-) )
1.7-2.0; δ stearylamine (-CH2NH-) ) 3.0-3.1; δ DL-pheny-
lanine (-CH2-) ) 3.1-3.4; δ GBA (-CH2NHC(dNH)NH-) )
3.3-3.4; δ lysine (-COCH(CH2CH2CH2CH2NH2)(NH-)) )
4.5-4.8; δ DL-phenylanine (-COCH(CH2C6H5)-) ) 4.7-5.0;
δ DL-phenylanine (-C6H5) ) 6.8-7.4.
Preparation of Nanoparticles. FITC-IILAVY loaded nano-
particles were prepared by the emulsion-solvent evaporation
technique (14). Briefly, the carrier (10 mg) dissolved in
methylene chloride (2 mL) was mixed with FITC-IILAVY (100
µg) dissolved in 500 µL DMSO. The mixture was slowly
dropped into poly(vinyl alcohol) aqueous solution (50 mL; 1.0%
w/v) while stirring at 3200 rpm. The pre-emulsion equilibrated
in an ice bath was sonicated for10 min by using a 750 W high-
intensity ultrasonic processor (VCX 750, Sonics and Materials
Inc., USA) operating at 20 kHz. The organic solvent was then
evaporated under gentle agitation (800 rpm) over 12 h at room
temperature. The resulting nanoparticles were dialyzed using
cellulose membrane (MWCO 3500 Da) in an excess amount of
water for 24 h and freeze-dried for 48 h to a powder form.
Particle Size Analysis. Size and size distribution of the drug-
loaded nanoparticles were measured by the particle size analyzer
(Zetasizer NanoZS2000; Malven Instrument, UK) based on
quasi-elastic light scattering. The nanoparticles were resuspended
at a 1.0 mg/mL (wt./v) concentration in deionized water. Then,
size and size distribution of the nanoparticles were measured
at room temperature (n ) 3).
Cytotoxicity Assay. After treating the BMMs with nanopar-
ticles for indicated time periods (12 h, 24 h, 36 h, and 48 h),
they were stained with propidium iodide (Sigma) and then
analyzed by a flow cytometer. The excitation wavelength was
488 nm.
RESULTS AND DISCUSSION
Synthesis. In order to enhance the cellular uptake of the
oligopeptides model drug into the BMMs, two series of dendritic
molecules with amine or guanidinium groups were synthesized
as shown in Figure 1. SF-A1 was synthesized by ring-opening
polymerization of N-carboxy anhydrides of DL-Phe on the
stearylamine. The degree of polymerization was about 3 by 1H
NMR spectrum. The SF-A2 was synthesized by the condensa-
tion reaction between carboxylic acid of R,ε-dibutyloxycarbonyl-
lysine and amino group of SF-A1 by using EDC and NHS as
coupling agents. After the deprotection of the butyloxycarbonyl
groups of SF-A2 by trifluoroacetic acid (TFA), SF-A2 with
dendritic amine groups was prepared. The repetition of the same
procedure gave dendritic SF-A4 with four amino groups.
To synthesize the SF-G1 with guanidinium groups, amino
group of SF-A1 was coupled with carboxylic acid of 4-guani-
dinobutyric acid (GBA) using EDC and NHS as coupling agents.
SF-G2 and SF-G4 were synthesized from SF-A2 and SF-A4,
respectively, by a similar coupling method. 1H NMR, IR
analysis, and MALDI-TOF mass spectra confirmed the structure
of SF-A1-SF-A4 and SF-G1-SF-G4. In 1H NMR spectra, the
methylene peak of the stearyl group at 1.2-1.4 ppm, methylene
peak of the lysine group at 1.7-1.8 ppm, and methylene peak
of the 4-guanidinobutyric group at 3.2 ppm confirm the progress
of the reaction (Supporting Information Figure S1a) (15, 16).
IR spectra of the SF-G1, SF-G2, and SF-G4 showed stronger
peaks at 1649 cm-1 and 1555 cm-1 corresponding to the
stretching vibration of CdN and bending vibration of N-H,
respectively (Supporting Information Figure S1b). The wide
band at 3400 cm-1 corresponds to the stretching vibration of
N-H group. The strong peaks at 1649, 1555, and 1380 cm-1
suggested that the guanidinylation had been successful (17).
MALDI-TOF mass spectra confirm the molecular ion peaks for
the products as indicated in the experimental section.
Determination of Drug Encapsulation Efficiency and
Drug Loading Content. The FITC-IILAVY loaded in the
nanoparticles was analyzed by HPLC (Waters LC). Briefly, the
drug loaded nanoparticles (1.0 mg) were dissolved in 1.0 mL
DMSO. A Waters Nova-Pak C18 (3.9ch150 mm, 4 µm) column
and UV/vis detector operating at 220 nm were used to analyze
the drug content in the nanoparticle. The flow rate of the mobile
phase (acetonitrile/water (50/50 by volume)) was 1.0 mL/min.
The drug encapsulation efficiency is defined by the amount of
drug encapsulated in the nanoparticle relative to the initial
amount of drug. The drug loading content is the ratio of the
amount of drug to nanoparticles.
Particle Analysis. During preparation of the FITC-IILAVY
loaded nanoparticles, the stearyl moieties act as the hydrophobic
core of the nanoparticles, whereas the amine groups or guani-
dinium groups with positive charges are located on the surface
of the nanoparticles. The poorly water-soluble FITC-IILAVY
was encapsulated into the polymeric nanoparticles due to
preferential partitioning of the drug by hydrophobic interactions.
The effective hydrodynamic size and zeta potential of FITC-
IILAVY loaded nanoparticles were estimated by using a
Cell Culture. BMMs were obtained from the femur of 5 week
old C57BL/6 mice. BMMs were flushed out of the bone marrow
cavity. BMMs were suspended for 3 days in a R-minimal
essential medium (R-MEM) (GIBCO-BRL, Grand Island, NY)
supplemented with 10% heat-inactivated fetal bovine serum, 100
units/mL penicillin, 100 µg/mL streptomycin (GIBCO), and 30

1476 Bioconjugate Chem., Vol. 21, No. 8, 2010
Chi et al.
Figure 2. Apparent size of SF-A2, SF-A4, SF-G2, and SF-G4 in water
determined by dynamic light scattering study.
Table 1. Zeta-Potential of the Carriers in Water
carrier
SF-A1 SF-A2 SF-A4 SF-G1 SF-G2 SF-G4
ꢀ potential (mv) 44 ( 3 54 ( 4 55 ( 1 52 ( 1 67 ( 1 85 ( 2
Zetasizer. SF-A1 and SF-G1 formed nanoparticles with a wide
range of 40-2000 nm and 50-500 nm, respectively, whereas
SF-A2, SF-A4, SF-G2, and SF-G4 formed nanoparticles with
a narrow range of 10-45 nm (Figure 2). The encapsulation
efficiency of the drug was 17-18% for SF-A2, SF-A4, SF-G2,
and SF-G4, resulting in the formation of nanoparticles with a
drug loading content of 0.45-0.55%. The drug encapsulated
nanoparticles were dialyzed using cellulose membrane (MWCO
3500 Da) in an excess amount of water for 24 h during which
some fraction of the drug might be released out of the
membrane, thus decreasing the encapsulation efficiency. When
the number of amine or guanidinium groups in the carrier
increased, size and size distribution of nanoparticles slightly
decreased. Zeta potential determines the surface charge of
nanoparticles. A high value of zeta potential indicates a high
surface charge of the nanoparticles, which leads to strong
repellent interactions among the nanopaticles in dispersion and
thus can give high stability to the nanoparticle in water. All
nanoparticles prepared from SF-A1-SF-A4 and SF-G1-SF-
G4 showed positive zeta potential in the range (44 ( 3)-(85
( 2) mV and good stability in water (Table 1). The zeta
potentials of the nanoparticles steadily increased as the number
of amine groups or guanidinium groups increased. In addition,
guanidinium modified carriers showed larger zeta potential than
amine modified carriers.
Cellular Uptake. Oligoarginine works as a cell penetrating
peptide and the effectiveness of cell penetration of oligoarginine-
conjugated protein (GCN 4) increased as the number of arginine
repeating units increased (10). Our system used the stearyl group
and oligophenylalanine as a hydrophobic block, where the drug
encapsulated nanoparticles are coated with guanidinium groups
by a self-assembly process. The cellular uptake of nanoparticles
containing FITC-IILAVY was investigated for SF-G4 and SF-
A4 as a function of time. The fluorescence intensity increased
with time (1 h, 1.5 h, 2 h, 3 h, 4 h) (Figure 3a). Two points can
be emphasized for our system. First, FACS analysis showed
that guanidinium groups modified nanoparticles (SF-G4) showed
excellent penetration into the BMMs. The oligoarginine with
four repeating units (R4) was reported to be ineffective as CPP
(10). However, current SF-G4 with four guanidinium groups
in a molecule showed excellent translocation ability. This
behavior comes from the self-assembly of the SF-G4 with
hydrophobic blocks of stearyl-oligophenylalanine and hydro-
philic blocks (G4). On a nanoparticle, the guanidinium groups
are located on the surface of the nanoparticle and exhibit a
polyarginine-like behavior. Second, guanidinium groups modi-
fied nanoparticles (SF-G4) were significantly more effective in
Figure 3. Intracellular delivery of oligopeptides into BMMs. Fluores-
cence images of internalized oligopeptides by SF-A4 (a) and SF-G4
(b). Both fluorescence images (left) and fluorescence/transmission
overlapped images (right) are shown. The scale bars in (a) and (b) are
10 µm. Flow cytometric histograms of internalized oligopeptides by
SF-A4 (c) and SF-G4 (d). Control experiment of the model drug is
carried out in the absence of carriers.
penetrating into the BMMs than amino group modified nano-
particles (SF-A4). The detailed mechanism of the difference is
under investigation. Previous study by Harashima et al. reported
that the octaarginine/DNA complex showed higher transfection
efficiency than octalysine/DNA complex (18). They suggested
that the enhanced transfection of octaarginine/DNA complex
comes from the different mode of endosomal escape from the
late endosome with low pH. In our system, the model neutral
oligopeptide drug was encapsulated in the guanidinium group
coated nanoparticle. In addition, the enhanced translocation of
the nanoparticle is observed more in the guanidinium group
coated nanoparticle than the amino group coated nanoparticle.
The favorable hydrogen bonding interactions between guani-
dinium groups with dispersed positive charges and negative
residues of the phospholipids present on the cell surface might
be responsible for such behavior (19, 20). Kinetic study also

Bioconjugate Chem., Vol. 21, No. 8, 2010 1477
Figure 4. Comparison of carriers for internalization of oligopeptides
into BMMs in 4 h. (a) Fluorescence images of internalized oligopeptides
by the functionalized carriers. Control experiment of the oligopeptides
is carried out in the absence of carriers. The scale bar is 10 µm. Both
fluorescence images (left) and fluorescence/transmission overlapped
images (right) are shown. (b) Flow cytometric histograms of internalized
oligopeptides by the functionalized carriers.
Figure 5. Cytotoxicity of carriers for BMMs. (a) FACS plot of BMMs
in 48 h of incubation. (b) Cell viability in the polymer solution (100
µg/mL in culture medium) for 48 h. Poly(L-lysine) (Mw ) 15 000-30 000
Da) was used for control.
suggested that 4 h is sufficient for the penetration study of the
nanoparticles for comparative purposes with other nanoparticles
of SF-A1-SF-A4 and SF-G1-SF-G4.
30 000) was used as a negative control in this research. SF-A4
and SF-G4 in a concentration of 100 µg/mL did not show
cytotoxicity, whereas more than 80% of the BMMs incubated
with PLL died.
To compare the penetration of the FITC-IILAVY loaded
nanoparticles into the BMMs, BMMs were incubated with
nanoparticles for 4 h. Fluorescence image and FACS analysis
suggested that the SF-G4 system most efficiently penetrates into
the cells (Figure 4). Many variables affect the uptake property
of nanoparticles, including adhesion to the cell surface, size,
and surface charge of the nanoparticle, and so forth. On the
basis of current results, we can speculate that the mechanisms
of internalization of nanoparticles through the cell membrane
may be endocytosis, where the positive charge of the nanopar-
ticles interacts with the negative charge of the phospholipid
membrane. SF-G4 nanoparticles possess higher density of
guanidinium group than SF-G1 and SF-G2. The higher uptake
of SF-G4 by BMM cells is caused by the high positive charge
of the nanoparticles as shown by a large ꢀ-potential. The fact
that SF-G4 showed higher translocation ability than SF-A4 also
supports previous suggestion that the guanidinium groups have
favorable interactions with negatively charged functional groups
in the cell membrane (19, 20). The resulting ion pairs have the
capability of translocation across the cell membrane.
Cytotoxicity Assay. A drug delivery system should possess
biocompatibility or cytocompatibility, as well as a delivery
function. HIV-Tat (48-60) was reported to show a 90% cell
survival rate at 100 µM for Hela cells and mouse macrophage
RAW264.7 (10, 21). We investigated the FACS plot of BMMs
after incubation for 48 h with SF-A4 and SF-G4 in the polymer
solution 100 µg/mL (62 µM for SF-G4 and 91 µM for SF-A4)
in culture medium, respectively (Figure 5). PLL (Mw 15 000-
To conclude, dendritic molecules with amine and guanidinium
groups were synthesized and their nanoparticles containing FITC
labeled model oligopeptide drug (FITC-IILAVY) were prepared.
FACS and spectrofluorimetry analysis showed that (1) the
efficiency of intracellular delivery increased by increasing the
number of the positive charged groups and (2) the nanoparticles
functionalized with dendrtic guanidinium groups demonstrated
better ability to cross the BMM membranes than amino groups.
The carriers (SF-A4 and SF-G4) did not exhibit any significant
cytotoxicity against BMMs. Current research suggests that SF-
G4 with hydrophobic block and four hydrophilic guanidinium
groups is a promising drug delivery carrier for peptide drug
targeting osteoclast precursors.
ACKNOWLEDGMENT
This work was supported by the Midcareer Researcher
Program through NRF grant funded by the MEST (2007-
0055790/2009-0080447) and Korea Science and Engineering
Foundation (KOSEF) National Research Laboratory (NRL)
Program grant funded by the Korean government (MEST)
(R0A-2008-000-20001-0).
Supporting Information Available: 1H NMR and FTIR of
all carriers (SF-A1-SF-A4, SF-G1-SF-G4) are provided. This
material is free of charge via the Internet at http://pubs.acs.org.

1478 Bioconjugate Chem., Vol. 21, No. 8, 2010
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