A novel poly(l -glutamic acid) dendrimer based drug delivery system with both pH-sensitive and targeting functions

Article abstract of DOI:10.1021/mp1000923

The functionalization of pH-sensitiveness and cellular targeting is a promising strategy to fabricate drug delivery systems with high efficiency, high selectivity and low toxicity. In this paper, a poly(l-glutamic acid) dendrimer based drug delivery system with both pH-sensitive and targeting functions is reported. Poly(l-glutamic acid) dendrimers with a polyhedral oligomeric silsesquioxane (POSS) nanocubic core were synthesized. Its globular morphology and compact structure with multiple peripheral functional groups made it suitable for drug delivery. The OAS-G₃-Glu dendrimer was conjugated with doxorubicin via pH-sensitive hydrazine bonds and targeting moiety (biotin). The cellular internalization and antitumor effects of the conjugates was evaluated in vitro. Both DLS and TEM results indicated that the conjugates aggregated into nanoparticles with diameters around 50 nm. The release rates of doxorubicin at pH 5.0 were much faster than those at pH 7.0 due to the acid cleavage of the hydrazine bonds. The internalization study revealed that the cellular uptake of the biotin modified conjugates was mainly through receptor-mediated endocytosis. These results indicate that our poly(l-glutamic acid) dendrimers with OAS core are promising vectors for fabricating smart and targeting drug delivery systems.

Full text of DOI:10.1021/mp1000923

articles
A Novel Poly(L-glutamic acid) Dendrimer Based Drug
Delivery System with Both pH-Sensitive and Targeting
Functions
Hui Yuan,† Kui Luo,† Yusi Lai, Yuji Pu, Bin He,* Gang Wang, Yao Wu, and
Zhongwei Gu*
National Engineering Research Center for Biomaterials, Sichuan UniVersity,
Chengdu 610064, China
Received April 1, 2010; Revised Manuscript Received May 16, 2010; Accepted May 19, 2010
Abstract: The functionalization of pH-sensitiveness and cellular targeting is a promising strategy
to fabricate drug delivery systems with high efficiency, high selectivity and low toxicity. In this
paper, a poly(L-glutamic acid) dendrimer based drug delivery system with both pH-sensitive
and targeting functions is reported. Poly(L-glutamic acid) dendrimers with a polyhedral oligomeric
silsesquioxane (POSS) nanocubic core were synthesized. Its globular morphology and compact
structure with multiple peripheral functional groups made it suitable for drug delivery. The OAS-
G3-Glu dendrimer was conjugated with doxorubicin via pH-sensitive hydrazine bonds and
targeting moiety (biotin). The cellular internalization and antitumor effects of the conjugates was
evaluated in vitro. Both DLS and TEM results indicated that the conjugates aggregated into
nanoparticles with diameters around 50 nm. The release rates of doxorubicin at pH 5.0 were
much faster than those at pH 7.0 due to the acid cleavage of the hydrazine bonds. The
internalization study revealed that the cellular uptake of the biotin modified conjugates was mainly
through receptor-mediated endocytosis. These results indicate that our poly(L-glutamic acid)
dendrimers with OAS core are promising vectors for fabricating smart and targeting drug delivery
systems.
Keywords: Poly(L-glutamic acid) dendrimer; targeting; drug delivery; doxorubicin; pH-sensitive
istics provide peptide dendrimers with excellent biodegrad-
ability and biocompatibility.5 These features have recently
been explored in the development of drug carriers. Most of
the drugs are immobilized on the peripheral functional groups
of peptide dendrimers to form dendrimer-drug conjugates.7
Introduction
Dendrimers offer many advantages as drug carriers for
their unique properties such as monodisperse, nanoscale size,
semiglobular or globular structures and multiple peripheral
functional groups. Extensive studies have already demon-
strated that dendrimers are good carriers for drugs in
transdermal, oral, ocular and pulmonary delivery systems.1-4
Peptide dendrimers originate from amino acids, and they
resemble protein-like globular structures. These character-
(2) Bai, S.; Thomas, C.; Ahsan, F. Dendrimers as a carrier for
pulmonary delivery of enoxaparin, a low-molecular weight
heparin. J. Pharm. Sci. 2007, 96 (8), 2090–2106.
(3) Yiyun, C.; Na, M.; Tongwen, X.; Rongqiang, F.; Xueyuan, W.;
Xiaomin, W.; Longping, W. Transdermal delivery of nonsteroidal
anti-inflammatory drugs mediated by polyamidoamine (PAMAM)
dendrimers. J. Pharm. Sci. 2007, 96 (3), 595–602.
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evaluation of dendrimer prodrugs for oral drug delivery. Int.
J. Pharm. 2007, 336 (1), 183–190.
* To whom correspondence should be addressed. B.H.: National
Engineering Research Center for Biomaterials, Sichuan Uni-
versity, 29# Wangjiang Road, Chengdu 610064, China; e-mail,
bhe@scu.edu.cn; tel, 86-28-85412923; fax, 86-28-85410653.
Z.G.: e-mail, zwgu@scu.edu.cn.
† Equally contributed to this paper.
(1) Vandamme, T. F.; Brobeck, L. Poly(amidoamine) dendrimers as
ophthalmic vehicles for ocular delivery of pilocarpine nitrate and
tropicamide. J. Controlled Release 2005, 102 (1), 23–38.
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Albericio, F. Peptide and amide bond-containing dendrimers.
Chem. ReV. 2005, 105 (5), 1663–1681.
10.1021/mp1000923  2010 American Chemical Society
Published on Web 05/19/2010
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The drugs are then released by the chemical or enzymatic
cleavage of the biodegradable labile bonds.6
and development. It must be obtained from exogenous
sources via intestinal absorption.20 The rapid proliferation
of cancer cells needs extra biotin, and the cancer cells often
overexpress biotin-specific receptors on the surface. Accord-
ingly, several research groups developed different biotiny-
lated chemotherapeutic agents for cancer cell-specific drug
delivery.21 The results have shown that biotin-conjugated
macromolecular carriers can enhance the uptake of anticancer
drugs to tumor cells.22
As the environment around tumor tissues is acidic, drug
carriers with pH-sensitive and targeting functions are desired
for antitumor drug delivery systems. There are many
advantages for peptide dendrimers as antitumor drug carriers.
The peripheral functional groups of peptide dendrimer can
not only immobilize targeting moieties but also link antitumor
drugs with pH-sensitive bonds. The peptide dendrimers thus
provide a useful platform for easy and convenient fabricati-
tion of smart and targeting drug delivery systems. In this
paper, we report the synthesis and characterization of poly(L-
glutamic acid) dendrimers with OAS cores. Doxorubicin
(DOX), a widely used antitumor drug, was conjugated to
the peripheral groups of the dendrimers through pH-sensitive
hydrazone bonds. In addition, biotin was immobilized on
the dendrimers as a specific tumor cell targeting moiety. Thus
the DOX-poly(L-glutamic acid) dendrimer conjugates were
fabricated with dual targeting and pH-sensitive functions.
We also report the subsequent studies of drug release,
antitumor effects and the cellular internalization of the
conjugates.
Polyhedral oligomeric silsesquioxane (POSS) units are
three-dimensional, cubic shaped building blocks that contain
an inorganic inner siloxane nanocore with the possibility of
chemical modification at each of the eight corners of the
POSS unit.7 The introduction of POSS core in peptide
dendrimers would largely amplify the amount of peripheral
groups and thus simplify the difficult synthetic process. Lu’s
group prepared poly(L-lysine) dendrimer with octa(3-ami-
nopropyl) silsesquioxane (OAS) as the cubic core and used
it as gene delivery vector.8 The dendrimers possessed a
globular morphology, a relatively rigid structure and a highly
functionalizable surface,9,10 which made OAS-cored den-
drimers ideal carriers for drug delivery systems.11
Nanodrug carriers could be concentrated in tumor cells
through the effect of enhanced permeability and retention
(EPR) or the mechanism of receptor-mediated endocytosis
(RME).12 The targeting delivery of high dose chemothera-
peutics using cancer cell-specific ligands is an attractive
alternative for the successful treatment of tumors.13,14 The
specific targeting moieties included sugar,15 folic acid,16
antibody,17 peptide18 and epidermal growth factor.19 Biotin
is an essential micronutrient for cellular functions, growth
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delivery processes. Chem. Commun. 2005, (8), 996–998.
(8) Kaneshiro, T. L.; Wang, X.; Lu, Z. R. Synthesis, characterization,
and gene delivery of Poly-L-lySine octa(3-aminopropyl)silses-
quioxane dendrimers: nanoglobular drug carriers with precisely
defined molecular Architectures. Mol. Pharmaceutics 2007, 4 (5),
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Dendrimers: Molecular-Level Control of Size, Shape, Surface
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Matter. Angew. Chem., Int. Ed. Engl. 1990, 29 (2), 138–175.
(11) Kaneshiro, T. L.; Lu, Z. R. Targeted intracellular codelivery of
chemotherapeutics and nucleic acid with a well-defined dendrimer-
based nanoglobular carrier. Biomaterials 2009, 30 (29), 5660–
5666.
(12) Tanaka, T.; Shiramoto, S.; Miyashita, M.; Fujishima, Y.; Kaneo,
Y. Tumor targeting based on the effect of enhanced permeability
and retention (EPR) and the mechanism of receptor-mediated
endocytosis (RME). Int. J. Pharm. 2004, 277 (1-2), 39–61.
(13) Dobson, P. D.; Kell, D. B. Carrier-mediated cellular uptake of
pharmaceutical drugs: an exception or the rule. Nat. ReV. Drug
DiscoVery 2008, 7 (3), 205–220.
(16) Majoros, I. J.; Myc, A.; Thomas, T.; Mehta, C. B.; Baker, J. R.
PAMAM dendrimer-based multifunctional conjugate for cancer
therapy: Synthesis, characterization, and functionality. Biomac-
romolecules 2006, 7 (2), 572–579.
(17) Shukla, R.; Thomas, T. P.; Peters, J. L.; Desai, A. M.; Kukowska-
Latallo, J.; Patri, A. K.; Kotlyar, A.; Baker, J. R. HER2 specific
tumor targeting with dendrimer conjugated anti-HER2 mAb.
Bioconjugate Chem. 2006, 17 (5), 1109–1115.
(18) Shukla, R.; Thomas, T. P.; Peters, J.; Kotlyar, A.; Myc, A.; Baker,
J. R. Tumor angiogenic vasculature targeting with PAMAM
dendrimer-RGD conjugates. Chem. Commun. 2005, (46), 5739–
5741.
(19) Yang, W. L.; Barth, R. F.; Wu, G.; Bandyopadhyaya, A. K.;
Thirumamagal, B. T. S.; Tjarks, W.; Binns, P. J.; Riley, K.; Patel,
H.; Coderre, J. A.; Ciesielski, M. J.; Fenstermaker, R. A.
Boronated epidermal growth factor as a delivery agent for neutron
capture therapy of EGF receptor positive gliomas. Appl. Radiat.
Isot. 2004, 61 (5), 981–985.
(20) Yellepeddi, V. K.; Kumar, A.; Palakurthi, S. Biotinylated Poly(a-
mido)amine (PAMAM) Dendrimers as Carriers for Drug Delivery
to Ovarian Cancer Cells In Vitro. Anticancer Res. 2009, 29 (8),
2933–2943.
(21) Minko, T.; Paranjpe, P. V.; Qiu, B.; Lalloo, A.; Won, R.; Stein,
S.; Sinko, P. J. Enhancing the anticancer efficacy of camptothecin
using biotinylated poly(ethyleneglycol) conjugates in sensitive and
multidrug-resistant human ovarian carcinoma cells. Cancer
Chemother. Pharmacol. 2002, 50 (2), 143–150.
(22) Russell-Jones, G.; McTavish, K.; McEwan, J.; Rice, J.; Nowotnik,
D. Vitamin-mediated targeting as a potential mechanism to
increase drug uptake by tumours. J. Inorg. Biochem. 2004, 98
(10), 1625–1633.
(14) Kannan, R. Y.; Salacinski, H. J.; Butler, P. E.; Seifalian, A. M.
Polyhedral oligomeric silsesquioxane nanocomposites: The next
generation material for biomedical applications. Acc. Chem. Res.
2005, 38 (11), 879–884.
(15) Bhadra, D.; Yadav, A. K.; Bhadra, S.; Jain, N. K. Glycodendrim-
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in an ice bath. The mixture was stirred at room temperature
overnight. After the evaporation of the methanol in reduced
pressure, sticky oil was formed. Chloroform was added and
a white precipitation was collected via centrifugation and
Materials and Methods
Materials. Sulfo-NHS-LC-biotin and avidin/hydroxya-
zobenzene-2-carboxylic acid (HABA) reagents were obtained
from Sigma-Aldrich (Shanghai, China). (3-Aminopropyl)tri-
ethoxysilane, succinic anhydride, N,N-diisopropylethylamine
(DIPEA), trifluoroacetic acid (TFA), 4-(dimethylamino)py-
ridine (DMAP), 1-ethyl-3-[3-(dimethylamino)propyl]carbo-
diimide (EDC) and NR-di-t-OBut-L-glutamic acid (H-
Glu(OBut)-OBut· HCl) were purchased from Asta Tech
Pharmaceutical (Chengdu, China). DOX hydrochloride (DOX-
HCl) was purchased from Zhejiang Hisun Pharmaceutical.
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate (HBTU) and 1-hydroxybenzotriazole hydrate
(HOBt) were obtained from Highfine Biotech Co. Ltd.
(Jiangsu, China). All the organic solvents used in this study
were purified using the standard method and distilled before
use.
1
dried in vacuum to obtain a white powder (2.2 g, 80%). H
NMR (D2O, δ): 0.59 (br, 2H, -SiCH2), 1.44 (br, 2H,
-SiCH2CH2), 2.30 (br, 2H, -COCH2CH2-), 2.41 (br, 2H,
-CH2CH2COOH), 3.02 (br, 2H, -Si(CH2)2CH2). 13C NMR
((CD3)2SO, δ): 8.74, 22.48, 29.15, 29.98, 41.0, 170.92,
173.89. 29Si NMR (D2O, δ): 65.3. MALDI-TOF (m/z, [M +
H]+): 1680 (calculated), 1681.40 (observed).
Synthesis of OAS-Poly(L-glutamic acid) (OAS-Glu)
Dendrimers. The reaction was carried out similarly to the
synthesis of poly-L-lysine octa(3-aminopropyl)silsesquioxane
dendrimers8 with some modifications. Generations 1, 2, and
3 of the dendrimers were simplified as G1, G2 and G3.
Synthesis of tert-Butyl Ester-Protected OAS-Poly(L-
glutamic acid) Dendrimers. Generation 0 (single OAS core)
to generation 2 (G0-G2) carboxyl terminated OAS-poly(L-
glutamic acid) dendrimers (1-2 g), HBTU (1.5 equiv with
respect to the carboxyl group), and HOBT (1.5 equiv with
respect to the carboxyl group) were dissolved in 30 mL of
anhydrous DMF in a three-necked flask equipped with a drop
funnel. Excess H-Glu(OBut)-OBut · HCl (2-3 equiv with
respect to the carboxyl group) was dissolved in 15 mL of
DMF and added dropwise. The solution was stirred at 0 °C
for 30 min in N2 atmosphere. N,N-Diisopropylethylamine (2
equiv with respect to the amount of H-Glu(OBut)-OBut ·HCl,
usually 3 mL) was added. The mixture was stirred at room
temperature for 2 or 3 days. After the removal of DMF,
chloroform was added and the mixture was washed with
saturated NaHCO3, NaHSO4, and brine solution several times.
The solution was dried with MgSO4 and the chloroform was
removed to give yellow solid. The crude product was recrystal-
lized in cold acetonitrile, and a colorless solid was obtained.
Deprotection of tert-Butyl Ester. A series of tert-butyl
ester-terminated OAS-poly(L-glutamic acid) dendrimers
(G1p-G3p) was treated with TFA (10 equiv, according to
the number of tert-butyl ester groups) in an ice bath, and
the mixture was stirred for 8-10 h to remove tert-butyl
groups. The solution was concentrated to give viscous oil.
The residue was treated with anhydrous diethyl ether, and a
colorless solid product was obtained. The yields of the
products were 90%, 85%, 75%, respectively.
1
Methods. H and 13C NMR spectra were recorded on a
Bruker Avance II NMR spectrometer at 400 MHz using
CDCl3 and DMSO-d6 as solvents with 0.5% tetramethylsilane
as the internal standard. The molecular weights of the
dendrimers were tested on Autoflex MALDI TOF MS
(Bruker, USA) in the linear mode with R-cyano-4-hydroxy-
cinnamic acid as a matrix. The absorbance of DOX with
different concentration was recorded on a Lambda 650S
UV-vis spectrometer (Perkin-Elmer) at 485 nm. The size
of particles was measured by dynamic light scattering (DLS)
(NANO ZSPO, Malvern) in aqueous solution. The morphol-
ogy of the particles was performed on a transmission electron
microscope (TEM) (H-600 electron microscope) with the
sample stained by phosphotungstic acid on a carbon-coated
copper grid. The laser confocal images were captured on a
LEICA TCS SP5 using a 40× water-immersion objective
lens and processed by Leica Confocal Software. DOX was
excited at 488 nm with emissions at 595 nm.
Synthesis of OAS · HCl. Octa(3-aminopropyl)silsesqui-
oxane hydrochloride (OAS ·HCl) was prepared according to
the previously reported method.23 Briefly, (3-aminopropyl)-
triethoxysilane (15 mL, 60 mmol) and 30%-36% concen-
trated HCl (30 mL) were dissolved in MeOH (350 mL) with
stirring for 3 days at room temperature. The crude product
was filtrated, washed with cold MeOH, and dried. The
product was recrystallized in hot MeOH to give white solid
1
(yield: 30%). H NMR ((CD3)2SO, δ): 8.23 (s, 24H), 2.76
Synthesis of tert-Butyl Hydrazinecarboxylate. tert-Butyl
Hydrazinecarboxylate(BocNHNH2) was synthesized accord-
ing to a published method.25 Briefly, hydrazine monohyate
(18 g, 80%) was mixed with isopropanol (300 mL), and a
solution of Boc2O (28 g, 128 mmol) in isopropanol (100
mL) was added dropwise at 0 °C for 2 h. Then the mixture
(t, 16H, -Si(CH2)2CH2), 1.71 (m, 16H, -SiCH2CH2), 0.72
(t, 16H, -SiCH2). 13C NMR ((CD3)2SO) δ: 40.53, 20.13,
7.96. 29SiNMR ((CD3)2SO,δ): 66.4. MALDI-TOF (m/z,
[M + H]+): 881.50 (calculated), 881.27 (observed).
Synthesis of OAS-SA. Succinic acid terminated OAS
(OAS-SA) was synthesized according to the published
method.24 Triethylamine (2 mL, 14 mmol) and succinic
anhydride (3.0 g, 3 mmol) were added orderly into a
methanol (150 mL) solution of OAS · HCl (2.0 g, 1.8 mmol)
(24) Tanaka, K.; Inafuku, K.; Adachi, S.; Chujo, Y. Tuning of
properties of POSS-condensed water-soluble network polymers
by modulating the cross-linking ratio between POSS. Macromol-
ecules 2009, 42 (10), 3489–3492.
(23) Gravel, M. C.; Zhang, C.; Dinderman, M.; Laine, R. M. Octa(3-
chloroammoniumpropyl) octasilsesquioxane. Appl. Organomet.
Chem. 1999, 13 (4), 329–336.
(25) Bredihhin, A.; Maeorg, U. Effective strategy for the systematic
synthesis of hydrazine derivatives. Tetrahedron 2008, 64 (28),
6788–6793.
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was stirred at room temperature overnight. After the isopro-
panol was evaporated, the residue was dissolved in CH2Cl2 and
dried over MgSO4. CH2Cl2 was removed, the product was
recrystallized in hexane, and colorless crystals were obtained.
1H NMR (CDCl3, δ): 6.13 (s, 1H, -CONHNH2), 3.68 (s, 2H,
-NH2), 1.42 (s, 9H, (CH3)3C-). 13C NMR (CDCl3, δ): 58.3,
77.2, 28.5.
Drug Release Experiment. The release studies were
performed at 37 °C in the phosphate buffer with different
pH values (pH 5.0 and 7.4). The OAS-G3-Glu(NHN-DOX)/
biotin complex (3c, 4 mg) was dispersed in 2 mL of PBS
medium and placed in a dialysis bag with a molecular weight
cutoff of 3 kDa. The dialysis bag was then immersed in 30
mL of the release medium and kept in a horizontal shaker
maintained at 37 °C for 170 rpm. 2 mL of the medium was
removed periodically, and the same volume of a fresh PBS
was added. The amount of released DOX was determined
with UV-vis spectrophotometry at 485 nm.
Cell Cytotoxicity. HeLa cells were cocultured with
dendrimers (compounds 3a and 3b) with different concentra-
tions to study cell cytotoxicity. The medium was DMEM
(Hyclone, Beijing) containing 10% fetal bovine serum and
1% antibiotics (antibiotic-antimycotic, Gibco BRL, USA) at
37 °C with 5% CO2 atmosphere. Cell cytotoxicity was tested
by Cell Counting Kit-8 assay.
In Vitro Antitumor Effects. The antitumor effects of
OAS-G3-Glu(NHN-DOX) and OAS-G3-Glu(NHN-DOX)/
biotin were evaluated in vitro with HeLa cells. The assay
was performed in triplicate with a density of 1 × 104 cells
per well in 96-well plates. After 48 h cultivation, the medium
was replaced with a fresh culture solution containing free
DOX, OAS-G3-Glu(NHN-DOX), and OAS-G3-Glu(NHN-
DOX)/biotin (the DOX content ranging from 3 µg/mL to
15 µg/mL) and incubated for 48 h. Thereafter, 10 µL of
CCK-8 was added to each well and incubated for another
1 h and the absorbance of each well was read on a scanning
enzyme-linked immunosorbent assay (ELISA) reader at a
wavelength of 490 nm.
Cellular Internalization Study. The cellular uptake was
examined on flow cytometry (Cytomics FC500, Beckman
Coulter) and confocal laser scanning microscopy. In flow
cytometry analysis, HeLa cells (about 2 × 105) were incubated
in six-well plates overnight. Three samples OAS-G3-Glu(NHN-
DOX), OAS-G3-Glu(NHN-DOX)/biotin (DOX concentration
was 10 µg/mL), and OAS-G3-Glu(NHN-DOX)/biotin with free
biotin (0.5 mg/mL) were added. After 2 h treatment, the cells
were rinsed with PBS buffer, trypsinized and resuspended in
500 µL of PBS buffer. The FACS Calibur flow cytometer was
used and analyzed approximately 104 cells from each sample.
The mean fluorescence of the gated viable cells was quantified
and shown on a four-decade log scale.
Synthesis of Compound 3a. The G3 · TFA (1 g, 0.12
mmol) was dissolved in DMF (30 mL). EDCI (4.2 g, 0.022
mol), HOBt (2.9 g, 0.022 mol) and DIEA (3 mL) were
added. The whole solution was activated at 0 °C for a
half-hour. Excess tert-butyl hydrazinecarboxylate (4.8 g,
0.037 mol) was added to the solution and stirred for a
further 2 days in nitrogen atmosphere. After removal of
the solvent, chloroform was added and the solution was
washed with saturated NaHCO3 and NaHSO4 solutions in
order. The solution was dried with MgSO4. The crude
product of OAS-G3-Glu-NHNHBOC was recrystallized in
acetonitrile, and a white solid was obtained (789 mg,
45%). 1H NMR ((CD3)2SO, δ): 10.17-9.30 (m, 64H,
-OCONHNH-), 9.06-8.44 (m, 64H, -NHNHCO-),
8.4-7.67 (m, 56H, -CONHCH- and 8H, -CH2NHCO-),
4.26 (s, 56H, -CH), 3.00 (s, 16H, -Si(CH2)2CH2), 2.45-2.00
(m, 32H, -COCH2CH2CO- and 144H, -CH2CH2CO-),
1.98-1.61 (d, 144H, -CHCH2CH2-), 1.57-1.15 (m, 592H,
(CH3)3C- and 16H, -SiCH2CH2), 0.58 (s, 16H, -SiCH2).
13C NMR ((CD3)2SO, δ): 179.02-168.45, 167.02-163.47,
163.93-160.90,159.01-153.12,103.61-94.92,79.53,57.19-46.84,
38.69, 36.23, 31.23, 28.50. MALDI-TOF (m/z, [M + H]+):
16217.7928 (calculated for C656H1128N192O268Si8), 16245.57
(observed). OAS-G3-Glu-NHNHBOC (500 mg) was treated
with TFA (2 mL) and precipitated in diethyl ether, resulting
in compound 3a as white powder (70%, 240 mg).
Synthesis of Compound (3b). OAS-G3-Glu-NHNH2 (3a,
500 mg) was dissolved in 4 mL of distilled water. Sulfo-NHS-
LC-biotin (370 mg) was added at a molar ratio of 1:20. The
mixture was reacted for 4 h and transferred to a dialysis bag
(MWCO ) 7000 Da) to remove the unreacted sulfo-NHS-LC-
biotin. The obtained liquid was lyophilized and stored.
4-Hydroxyazobenzene-2-carboxylic Acid (HABA)
Assay. 4-Hydroxyazobenzene-2-carboxylic acid (HABA)
/AVIDIN REAGENT (Sigma, product No. H 2153) was used
to define the content of biotin on each dendrimer. The reagent
was prepared according to the manufacturer’s instructions
by simple reconstitution with 10 mL of deionized water.
Briefly, 100 µL of biotinylated dendrimer solution was added
to 900 µL of the avidin/HABA reagent solution and the
absorbance was measured at 500 nm using UV-vis
spectrometry.
Formation of DOX-Dendrimer Conjugates (3c). OAS-
G3-Glu-NHNH2/biotin (3b, 12 mg) and excess DOX (300
mg) were dissolved in 50 mL of anhydrous methanol with a
drop of acetic acid as a catalyst. The solution was stirred at
room temperature in the dark for 40 h. The product solution
was ultrafiltered and washed in Amicon Ultra tubes. The
unreacted DOX was further removed with a Sephadex LH-
20 column.
For confocal microscopy studies (CLSM), HeLa cells were
seeded on 22 mm glass coverslips in a six-well plate on the
day before the experiment. The cells were treated with free
DOX, OAS-G3-Glu(NHN-DOX), and OAS-G3-Glu(NHN-
DOX)/biotin (those three with the same DOX concentration:
10 µg/mL) for 0.5 and 2 h, washed and fixed with 1.5%
formaldehyde and watched under confocal laser scanning
microscopy.
Results and Discussion
The synthetic process of OAS-poly(L-glutamic acid)
dendrimers is shown in Scheme 1. With the consideration
of the instability of the silsesquioxane units in aqueous
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Scheme 1. The Formation of OAS-Poly(L-glutamic acid) Dendrimers
alkaline solutions26 and the incomplete hydrolysis of methyl
or ethyl ester under acidic catalysts,27 tert-butyl ester-
protected glutamic acid was used as the reagent. An excess
of NR-di-t-OBut-L-glutamic acid · HCl (H-Glu(OBut)-
OBut· HCl) was used in each step to ensure the complete
reaction of the surface carboxyl groups with tert-butyl ester-
protected -glutamic acid.
L
(26) Feher, F. J.; Wyndham, K. D.; Soulivong, D.; Nguyen, F.
Syntheses of highly functionalized cube-octameric polyhedral
oligosilsesquioxanes (R8Si8O12). J. Chem. Soc., Dalton Trans.
1999, (9), 1491–1497.
(27) Naka, K.; Fujita, M.; Tanaka, K.; Chujo, Y. Water-soluble anionic
POSS-Core dendrimer: Synthesis and copper(II) complexes in
aqueous solution. Langmuir 2007, 23 (17), 9057–9063.
The formation OAS-poly(L-glutamic acid) dendrimers with
tert-butyl ester groups was confirmed by 1H NMR as shown
in Figure 1. The peaks at 0.54 ppm, 1.47 ppm and 3.02 ppm
were the characteristic R-, ꢀ- and γ-protons of the methylene
groups adjacent to the silicon atoms of the OAS cores,
consistent with the results previously reported.8 The signal
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Figure 2. The 1H NMR spectra of (a) OAS-G3-Glu-
NHNHBoc and (b) OAS-G3-Glu-NHNH2/biotin.
spectra. The double peaks at 8.6-9.7 ppm were attributed
to the protons of newly formed bond (-NHNH-).
Biotin is a member of vitamin family (vitamin B) and is
an essential micronutrient whose level is high in rapidly
proliferating cells such as cancer cells.30 Several research
groups have demonstrated that dendrimers with biotin
molecules contribute to the enhancement of specific cancer
cell uptake.22,31-33 Sulfo-NHS-LC-biotin, a derivative of
D-biotin, acted as a biotinylation agent. The most important
advantage of this reagent was that the 6-aminocaproic acid
spacer prolonged the length between the covalently modified
molecule and the bicyclic biotin rings,20 which sharply
reduced steric hindrance and improved drug loading efficiency.
Figure 1. The 1H NMR spectra of tert-butyl ester-protected
OAS-poly(L-glutamic acid) dendrimers.
at 1.38 ppm was attributed to the protons of tert-butyl group
in the glutamic acid skeleton. The peaks at 2.05-2.42 ppm
were ascribed to the γ-protons of the L-glutamic acid side
chains and the methylene groups in the succinic anhydride
chains. The presence of two multiples at 1.70 and 1.86 ppm
was assigned to the two methylene protons next to the
R-proton of the peptide bond, and their integrity ratio was
After the removal of Boc groups, the resultant OAS-G3-
Glu-NHNH2 dendrimers were reacted with sulfo-NHS-LC-
1
1:1. The peak at 4.10 ppm in the H NMR spectrum of G1
was assigned to the R-proton of glutamic acid. When a
second generation of L-glutamic acid was conjugated to the
dendrimer surface, this peak split into two peaks between
4.09 and 4.23 ppm because of the different chemical
environment after the conjugation to the R- and γ-carboxyl
groups in the L-glutamic residue of G1 dendrimers. The peaks
at 7.71-8.24 ppm were observed due to the active proton
in the formed amide bond.
(28) Kono, K.; Kojima, C.; Hayashi, N.; Nishisaka, E.; Kiura, K.;
Wataral, S.; Harada, A. Preparation and cytotoxic activity of
poly(ethylene glycol)-modified poly(amidoamine) dendrimers
bearing adriamycin. Biomaterials 2008, 29 (11), 1664–1675.
(29) Kono, K.; Liu, M. J.; Frechet, J. M. J. Design of dendritic
macromolecules containing folate or methotrexate residues. Bio-
conjugate Chem. 1999, 10 (6), 1115–1121.
(30) Yang, W. J.; Cheng, Y. Y.; Xu, T. W.; Wang, X. Y.; Wen, L. P.
Targeting cancer cells with biotin-dendrimer conjugates. Eur.
J. Med. Chem. 2009, 44 (2), 862–868.
(31) Marek, M.; Kaiser, K.; Gruber, H. J. Biotin-pyrene conjugates
with poly(ethylene glycol) spacers are convenient fluorescent
probes for avidin and streptavidin. Bioconjugate Chem. 1997, 8
(4), 560–566.
(32) Cannizzaro, S. M.; Padera, R. F.; Langer, R.; Rogers, R. A.; Black,
F. E.; Davies, M. C.; Tendler, S. J. B.; Shakesheff, K. M. A novel
biotinylated degradable polymer for cell-interactive applications.
Biotechnol. Bioeng. 1998, 58 (5), 529–535.
In order to fabricate pH sensitive drug delivery system,
the tert-butyl ester-protected OAS-G3-Glu dendrimer was
deprotected and substituted with tert-butyl hydrazinecar-
boxylate to immobilize DOX with hydrazide bond. If
hydrazine hydrate was used in usual conditions,28,29 the
silsesquioxane units would break down in the strong basic
environment and an intermolecular reaction among dendrim-
ers would occur. So, in this paper, tert-butyl hydrazinecar-
boxylate was used to avoid this problem. The obtained OAS-
1
G3-Glu-NHNHBOC dendrimer was confirmed by H NMR
(33) Na, K.; Lee, T. B.; Park, K. H.; Shin, E. K.; Lee, Y. B.; Cho,
H. K. Self-assembled nanoparticles of hydrophobically-modified
polysaccharide bearing vitamin H as a targeted anti-cancer drug
delivery system. Eur. J. Pharm. Sci. 2003, 18 (2), 165–173.
spectra as shown in Figure 2a. With the comparison of the
1H NMR spectra of OAS-G3-Glu dendrimer, the character-
istic peaks of the OAS core were both observed in the two
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Scheme 2. The Formation of OAS-G3-Glu(NHN-DOX)/Biotin Complex
Table 1. The Composition of DOX-Dendrimer Conjugates
biotin molecules (1:20). The peaks between 6.3 and 6.5 ppm
in the 1H NMR spectrum were the characteristic protons of
biotin as shown in Figure 2b. The content of biotinylation
was calculated using the formula given by Sigma-Aldrich
(the product information of HABA/AVIDIN REAGENT).
terminal
drug
sample
group [DOX] [biotin] MWa loading (%)
G3-Glu(NHNH2)
G3-[DOX]
64
56
54
9835
14131
14026
8
6
30
24
G3-[DOX]-[biotin]
4
( )
1
µmol/mL ) 10 ∆A /34
( )
500
a Molecular weights calculated from its structure.
34 ) mM extinction coefficient at 500 nm
10 ) dilution factor of sample into cuvette
Doxorubicin molecules were bonded to both biotinylated
(OAS-G3-Glu-NHNH2/biotin) and biotin-free (OAS-G3-Glu-
NHNH2) agents through Schiff bases as shown in Scheme
2. The composition of the conjugates is shown in Table 1.
( )
2
∆A
) 0.9A
)
- A
HABA/avidin HABA/avidin+sample
(
(
)
500
0.9 )
Particle size and distribution are two important aspects in
determining whether the conjugates are suitable for drug
delivery. It was proposed that nanoparticles (NPs) with sizes
less than 5 µm would be taken up via the lymphatics and
sizes less than 500 nm could cross the membranes of
dilution factor of HABA/avidin upon addition of sample
The results of both 1H NMR (Figure 2b) and avidin/HABA
assay revealed that four biotin molecules were in average
conjugated to each dendrimer.
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Figure 3. Dynamic laser scattering (DLS) of OAS-G3-
Glu(NHN-DOX)/biotin complexes.
Figure 5. In vitro DOX releases from Glu(NHN-DOX)/
biotin complex at pH 7.4 and 5.0.
Figure 4. TEM image of OAS-G3-Glu(NHN-DOX)/biotin
complexes.
epithelial cells through endocytosis.34,35 The size distribution
of the OAS-G3-Glu(NHN-DOX)/biotin complexes was in-
vestigated by DLS. Figure 3 shows the size distribution of
the complex at a concentration of 10 µg/mL. As shown in
Figure 3, the size distribution of the complex is rather narrow
and the average hydrodynamic diameter is peaked at 45 nm.
This size range makes these particulate conjugates ideal
candidates for drug delivery as far as size is concerned.
The size and morphology of the OAS-G3-Glu(NHN-
DOX)/biotin conjugates were further evaluated by TEM.
Figure 4 shows that the nanoparticles are spherical and
uniform. The diameters of the complexes as observed by
TEM are in the range of 30-50 nm, consistent with the size
range from the DLS studies. These sizes are slightly bigger
than the individual dendritic conjugate molecules themselves,
and the difference is clearly due to the conjugate aggregation.
The nanoparticles with diameters less than 100 nm were
suitable for injectable anticancer drug carriers due to the EPR
effect.36 Hence, these nanoparticles aggregated from the
conjugates are adequate for antitumor drug delivery.
Figure 6. Cytotoxicity of drug carriers: (a) OAS-G3-NHNH2
and (b) OAS-G3-NHNH2/biotin against HeLa cell after
48 h incubation.
The release behavior of DOX from dendrimer-drug
conjugates under different pH conditions was investigated
to evaluate how pH variation affected the cleavage of
polymer-drug linkages. Figure 5 shows the release of DOX.
DOX release was found to be slow at pH 7.4, and the
percentage of the released DOX was only 12% within 24 h,
showing that the hydrazone bond was relatively stable at pH
7.4. On the contrary, a much faster release of DOX was
observed at pH 5.0. Clearly, the cleavage of the pH sensitive
hydrazone bonds became far easier in slightly acidic
environment. After 20 h, the totally released DOX reached
about 90% at pH 5.0.
(34) Mu, L.; Feng, S. S. Vitamin E TPGS used as emulsifier in the
solvent evaporation/extraction technique for fabrication of poly-
meric nanospheres for controlled release of paclitaxel (Taxol).
J. Controlled Release 2002, 80 (1-3), 129–144.
(35) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Micellar
nanocontainers distribute to defined cytoplasmic organelles.
Science 2003, 300 (5619), 615–618.
(36) Yuan, F.; Dellian, M.; Fukumura, D.; Leunig, M.; Berk, D. A.;
Torchilin, V. P.; Jain, R. K. Vascular permeability in a human
tumor xenograft: molecular size dependence and cutoff size.
Cancer Res. 1995, 55 (17), 3752–3756.
The cytotoxicity of the dendritic carriers was evaluated.
As indicated in Figure 6, the cytoxicity of the biotin free
and biotin modified dendrimers is closely related to its
concentrations. Both dendrimers had low cytotoxicity when
the concentrations were below 15 µg/mL. The cytotoxicity
then increased with the increase of dendrimer concentration.
The cytotoxicity was likely to originate from the cationic
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Figure 8. Flow cytometry results of HeLa cells that were
incubated with OAS-G3-Glu(NHN-DOX), OAS-G3-Glu(NHN-
DOX)/biotin (DOX concentration is 10 µg/mL) and OAS-
G3-Glu(NHN-DOX)/biotin (in the presence of 500 µg/mL
free biotin) for 2 h at 37 °C.
Figure 7. The cytotoxicity of free DOX, OAS-G3-Glu(NHN-
DOX), and OAS-G3-Glu(NHN-DOX)/biotin against HeLa
cells after 48 h incubation. DOX concentrations were 3,
6, 9, 12, 15 µg/mL for each group.
interaction between positively charged conjugates and the
negatively charged cell membranes, the OAS-G3-Glu(NHN-
DOX) showed a normal dose response after 2 h incubation.
Although the DOX concentration and cell incubation time
of OAS-G3-Glu(NHN-DOX) and OAS-G3-Glu(NHN-DOX)/
biotin were the same, the fluorescence intensity of OAS-G3-
Glu(NHN-DOX)/biotin exhibited was higher than that of
OAS-G3-Glu(NHN-DOX). This difference clearly arose from
the biotin facilitation of the cell uptake process. To further
determine the role of biotin in cell specific uptake process,
the fluorescence intensity of the OAS-G3-Glu(NHN-DOX)/
biotin conjugates (DOX concentration was kept the same as
the above two samples) with additional free biotin (0.5 mg/
mL) was tested. Interestingly, the presence of excess biotin
effectively decreased the cellular uptake of OAS-G3-Glu(NHN-
DOX)/biotin conjugates to the HeLa cells, with a reduction
of nearly 30% of the cell fluorescence intensity being
observed. This funding was consistent with the previous
studies,30 though the exact mechanistic process causing the
apparent “overdosing” remains unclear. Nevertheless, these
results together show that the cellular uptake of DOX-
dendrimer conjugates can be enhanced with the introduction
of biotin and that the OAS-G3-Glu(NHN-DOX)/biotin con-
jugates must be mainly internalized by the biotin-receptor
mediated endocytosis.
charges37 of the dendrimers. It was lower in biotin-im-
mobilized dendrimers because the bond linkages reduced the
level of cationic charges.
Figure7showstheantitumoreffectsoftheDOX-dendrimer
conjugates. The viability of HeLa cells incubated with free
DOX, OAS-G3-Glu(NHN-DOX) and OAS-G3-Glu(NHN-
DOX)/biotin (DOX concentrations ranged from 3 µg/mL to
15 µg/mL) for 48 h were tested. Cell viability dramatically
decreased in the presence of free DOX, and it maintained at
4%-8% even though the concentration of DOX increased,
showing that the DOX concentration of 3 µg/mL was enough
to kill tumor cells in vitro. The DOX released from
DOX-dendrimer conjugates exhibited lower antitumor ef-
fects. When the concentration was higher than 6 µg/mL, the
cell viability of conjugates with biotin was much lower than
that of conjugates without biotin. The difference demon-
strated that the targeting moiety offered by biotin was
efficient at enhancing the tumor cell targeting in vitro.
The cell uptake and intracellular distribution of the
conjugates were studied by flow cytometry analysis and
confocal laser scanning microscopy (CLSM). Flow cytometry
analysis was carried out to quantitatively evaluate the
endocytosis of OAS-G3-Glu(NHN-DOX) and OAS-G3-
Glu(NHN-DOX)/biotin (DOX concentration was kept at 10
µg/mL). Since DOX itself is a fluorophore, the fluorescence
intensity is proportional to the amount of internalized DOX
in Hela cells. For the results shown in Figure 8, we have
used the cells without any treatment as control. These cells
showed autofluorescence only. Due to the nonspecific
Confocal laser scanning microscopy (CLSM) was used to
visualize the internalization of the conjugates. Figure 9 shows
the confocal images of HeLa cells treated with free DOX,
OAS-G3-Glu(NHN-DOX) and OAS-G3-Glu(NHN-DOX)/
biotin for 0.5 and 2 h. In each time, the cells exposed to the
free DOX showed the highest fluorescence intensity in the
nuclei, due to the rapid transport of DOX into the cytosol,
followed by the subsequent diffusion to nuclei and aggrega-
tion there.38 The fluorescence intensity of OAS-G3-Glu(NHN-
DOX)/biotin was much stronger than that of OAS-G3-
(37) Malik, N.; Wiwattanapatapee, R.; Klopsch, R.; Lorenz, K.; Frey,
H.; Weener, J. W.; Meijer, E. W.; Paulus, W.; Duncan, R. Erratum
to “Dendrimers: Relationship between structure and biocompat-
ibility in vitro, and preliminary studies on the biodistribution of
125I-labelled polyamidoamine dendrimers in vivo” [Journal of
Controlled Release 65 (2000) 133-148]. J. Controlled Release
2000, 68 (2), 299–302.
(38) Gillies, E. R.; Frechet, J. M. J. pH-responsive copolymer
assemblies for controlled release of doxorubicin. Bioconjugate
Chem. 2005, 16 (2), 361–368.
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Figure 9. CLSM images of HeLa cells. The cells were incubated with (a) free DOX, (b) OAS-G3-Glu(NHN-DOX) and
(c) OAS-G3-Glu(NHN-DOX)/biotin for 0.5 h and 2 h.
Glu(NHN-DOX), consistent with the enhancement of cellular
internalization associated with the biotin’s targeting effect
via the receptor-mediated endocytosis. In contrast, the biotin-
free conjugates were taken up by the HeLa cells through
the simple endocytosis process. The amount of conjugates
internalized was less, resulting in the weaker fluorescence
intensity of OAS-G3-Glu(NHN-DOX). The fluorescence
intensity of both OAS-G3-Glu(NHN-DOX) and OAS-G3-
Glu(NHN-DOX)/biotin increased with time, and this effect
was attributed to the cleavage of the pH-sensitive bonds in
the acidic endosome. These results demonstrated the sus-
tained release of the DOX from the conjugates and that the
general trend was consistent with the previous observations
from other systems.39 But the dendritic and polypeptide
nature has made the current systems more attractive for
further exploration toward the development of cancer therapy.
doxorubicin was conjugated to the OAS-G3-glutamate den-
drimers via an acid-labile hydrazine linkage. Biotin was also
immobilized on the peripheral groups of the dendrimers as
targeting moiety. The diameters of the aggregated conjugates
were in the range of 30-50 nm. The conjugates with biotin
showed a pH-activated drug release feature and a much
higher cellular uptake compared to the conjugates without
biotin. The in vitro antitumor effects of the conjugates with
biotin, as studied from HeLa cell culturing, were much better
than those obtained from the conjugates without biotin. These
results have indicated that the introduction of both pH
sensitive bonds and targeting moiety to the OAS-G3-Glu
dendrimers with nanocubic structure can improve drug
delivery efficiency.
Acknowledgment. This research work was supported
by the National Basic Research Program of China (National
973 program, No. 2005 CB623903), National Science
Foundation of China (NSFC, 50633020).
Conclusion
Poly(L-glutamic acid) dendrimers with silsesquioxane core
1
were synthesized and characterized by H NMR, 13C NMR
Supporting Information Available: Detailed descriptions
of synthetic procedures for the dendrimers described here and
supporting mass spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
and MALDI-TOF mass spectrometry. The anticancer drug
(39) Ulbrich, K.; Subr, V. Polymeric anticancer drugs with pH-
controlled activation. AdV. Drug DeliVery ReV. 2004, 56 (7), 1023–
1050.
MP1000923
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