Heterobifunctional Poly(ethylene glycol) Derivatives for the Surface Modification of Gold Nanoparticles Toward Bone Mineral Targeting

Article abstract of DOI:10.1002/mabi.201200046

Heterobifunctional poly(ethylene glycol)s can be used for many biomedical applications ranging from solubility enhancement of hardly soluble compounds to surface modification of medical devices. In order to modify gold nanoparticles as model particles for

Full text of DOI:10.1002/mabi.201200046

Full Paper
Heterobifunctional Poly(ethylene glycol)
Derivatives for the Surface Modification
of Gold Nanoparticles Toward Bone
Mineral Targeting
Gamal M. S. Zayed, Joerg K. V. Tessmar*
Heterobifunctional poly(ethylene glycol)s can be used for many biomedical applications
ranging from solubility enhancement of hardly soluble compounds to surface modification
of medical devices. In order to modify gold nanoparticles as model particles for drug targeting
applications, PEG derivatives are synthesized that pos-
sess a high affinity for gold surfaces, namely a thioalkyl
function, known to form stable monolayers on gold.
Additionally a bisphosphonate function is introduced
in the PEG molecule to allow targeting of hydroxyapatite
rich tissues, like bone. Gold nanoparticles are modified
using the synthesized bifunctional PEG and investigated
for their stability in biological fluids and their ability to
bind to hydroxyapatite granules in these fluids.
1. Introduction
functions, like for example a controlled cellular adhesion,
immobilization of bioactive compounds, or also the
targeting to a certain cell type or tissue of interest in case
of modified nanoparticles.[4] The so far most prominent
applications of functionalized PEG molecules include:
PEGylation of peptide and protein drugs,[6–10] preparation
of hydrogels for drug delivery applications,[11–13] as well as
the surface modification of materials in order to specifically
control cellular adhesion[13,14] or the surface stabilization
and functionalization of nanoparticles.[15–17]
Inorder to functionally modifybiomaterialsurfaces such as
polymers or metals, derivatives of poly(ethylene glycol)
(PEG) play an important role due to their unique properties
such as high water solubility, good biocompatibility, low
cytotoxicity, low immunogenicity, low protein binding,
and subsequently poor cell adhesion.[1–3] An additionally
introduced second functionality into the polymer further-
more allows for a selective attachment to surfaces and the
presentation of specific groups on the modified surface.[4,5]
These modified surfaces can then exert further biological
For the creation of modified surfaces thiol functionalized
or thioalkylated PEGs are well established, as they are
known to bind strongly to various gold coated surfaces due
to the spontaneous and very specific adsorption of the thiol
or disulfide derivatives to gold, which ultimately leads to
the formation of often very stable polymer monolayers on
top of the gold.[18,19] For a successful modification of the
surface, an alkanethiol part within the polymer layer is
essential to form a highly stable, highly ordered and well
oriented monolayer film due to the additional presence of
Dr. G. M. S. Zayed, Dr. J. K. V. Tessmar
Department of Pharmaceutical Technology, University of
Regensburg, 93040 Regensburg, Germany
E-mail: joerg.tessmar@ur.de
Dr. G. M. S. Zayed
Current Address: Department of Pharmaceutical Technology,
Faculty of Pharmacy, Al-Azhar University, Assiut, Egypt
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acid, sodium acetate, ammonium carbonate, diethyl ether, hydro-
xyapatite (HA), trisodium citrate dihydrate, hydrochloric acid,
nitric acid, Endobone granules, sodium chloride, and calcium
chloride were purchased from Merck (Darmstadt, Germany).
Deuterated chloroform was from Deutero GmbH (Kastellaun,
Germany). Ibandronate as standard substance was a generous gift
from Boehringer (Mannheim, Germany).
van der Waals interactions between neighboring alkyl
chains, which almost form crystal structures during their
assembly.[20,21]
In order to create highly inert surfaces the anchoring part
of the alkane thiol has to be covalently linked to PEG as
surface coating, which iswell known to prevent non-specific
adsorption of proteins. The PEG mediated protein resistance
is due to the formation of stable interfacial aqueous layer,
which prevents a direct contact between the underlying
biomaterialsurfaceandtheproteinsinsolution,andexhibits
no further interactions (ionic or van der Waals interactions)
betweentheunchargedPEGandtheproteinmolecules.[22–24]
In order to synthesize accordingly functionalized PEG
molecules a strategy had to be developed, which allowed
introducing different functionalities in the PEG chains. For
this approach asymmetric monoamine-PEG was synthe-
sized and attached to the alkanethiol part as well as to a
bisphosphonic acid, which is known for its high binding
affinity to bone mineral, which served as model tissue.[25]
The main drawbacks for conjugation of clinically used
bisphosphonates (BPs), like alendronate of pamidronate, to
polymers are the difficulties to detect their binding due to
the lack of a suitable chromophore and furthermore their
limited solubility in non-aqueous media due to the high
polarity. Therefore, an already described BP derivative[26]
containing an aromatic ring was used to detect the
modification of the polymer and a protected BP was used
to ease the linkage of the molecules in organic solution.
The goal of this study was to establish the synthesis of a
BP labeled PEG, which is suitable for the surface functio-
nalization of differently sized gold particles. The modified
particles should be suited to investigate the targeting
ability of the attached BP ligand for differently sized model
nanoparticles to bone tissue.
All glassware was thoroughly washed with freshly prepared
aqua regia (HCl/HNO3 ¼ 3:1), extensively rinsed with Millipore
water several times and oven-dried at 150 8C for 2–3 h before use.
All used solutions filtered through a 0.22 mm filter before use.
2.1.1. Synthesis of 3,5-Di(tetraethylethylamino-2,2-
bisphosphonate)benzoic Acid
3,5-Di(tetraethylethylamino-2,2-bisphosphonate)benzoic acid as
bisphosphonate carrying label for the polymers was synthesized
according to a procedure described by Bansal et al.[26] (Figure 1).
Briefly, dietylamine (0.580 g, 8 mmol) and paraformaldeyde
(1.212 g, 40 mmol) were added to 25 mL of methanol and the
mixture was slightly warmed until a clear solution was obtained.
Tetraethyl methylenediphosphonate (1) (2.326 g, 8 mmol) was
added and the mixture was refluxed for 24 h, finally methanol was
removedunder vacuum and twice replaced with toluene and again
evaporated to ensure complete removal of methanol. Crude
intermediate was used to obtain a 1H NMR spectrum [dH (CDCl3):
4.19(m, 8H, OCH2CH3), 3.63(m, 2H,CH3OCH2), 3.2(s, 3H, CH3O), 2.52
(tt, 1H, PCHP), and 1.18 (t, 12H, CH2CH3)]. The resulting crude
product tetraethyl 2-methoxyethylene-diphosphonate (2) was
finally dissolved in 100 mL toluene and catalytic amounts of
p-toluenesulfonic acid monohydrate (5 mg) were added and the
solutionwasrefluxedfor14 husingaSoxhletapparatuscontaining
calcium hydride for the complete removal of methanol and water.
After the reaction toluene was evaporated and the obtained
product was dissolved in chloroform (50 mL) and washed three
times with 30 mL water. The organic layer was subsequently dried
with anhydrous sodium sulfate and concentrated to yield an oily
residue. The crude product was purified by fractional distillation to
a produce a clear liquid of the boiling point 115–116 8C, which is
characteristic for the tetraethylethenylidene bis(phosphonate)
intermediate (3) and an NMR spectrum was recorded [dH (CDCl3):
6.98 (dd, 2H, CH2¼), 4.15 (m, 8H, OCH2CH2), and 1.3 (m, 12H,
CH3CH2)].[27] The intermediate (3) (0.96 g, 3.2 mmol) and 3,5-
diaminobenzoic acid (4, 0.24 g, 1.6 mmol) were subsequently
heated in 30 mL of THF at 60 8C for 5 h to produce 3,5-
di(tetraethylethylamino-2,2-bisphosphonate)benzoic acid (5),
which was subsequently dried under vacuum and analyzed with
NMR [dH (CDCl3): 6.91 (s, 2H, ortho protons of benzene ring), 6.15 (s
1H, para proton of benzene ring), 4.15 (m, 16H, ꢁOꢁCH2ꢁCH3), 3.78
(m, 4H, ꢁNHꢁCH2ꢁCH), 2.75 (tt, 2H, PꢁCHꢁP), and 1,37 (t, 24H,
ꢁOꢁCH2ꢁCH3)].
2. Experimental Section
2.1. Materials
Tetraethylmethylene diphosphonate, diethylamine, 11-bromo-1-
undecene, sodium hydride, N-hydroxysuccinimide (NHS), N,N0-
dicyclohexylcarbodiimide (DCC), ethylene oxide, and ammonium
persulfate, were obtained from Fluka (Buchs, Switzerland).
p-Toluenesulfonic acid monohydrate, 3,5-diaminobenzoic acid,
PEG monomethyl ether (mPEG, Mw 2000 Da), a,a0-azoisobutyroni-
trile (AIBN), thioacetic acid, potassium bis(trimethylsilyl) amide,
di-t-butyl dicarbonate (BOC), tetramethylsilane (TMS), bromotri-
methylsilane, hydrogen tetrachloroaurate trihydrate (HAuCl4 ꢀ 3H2O),
bovine serum albumin (BSA), ultrafiltration tubes [molecular
weight cut-off (MWCO) 50 000 Da)], DEAE-sephadex A25 and
dialysis tubing with an MWCO of 1000 Da were acquired from
Sigma-Aldrich Chemical Company (Steinheim, Germany). Para-
formaldeyde, calcium hydride, methanol, toluene, dichloro-
methane, chloroform, acetonitrile, tetrahydrofuran (THF), acetone,
1,4-dioxane, sodium sulfate, hydrochloric acid, sulfuric acid, acetic
2.1.2. Synthesis of the PEG Undecylmercaptane
Derivatives
To synthesize thioalkylated PEG monomethyl ether, the synthetic
scheme in Figure 2 was applied. 5 g (2.5 mmol) of methoxy PEG (7a)
were melted in an oil bath under argon atmosphere. 600 mg
(25 mmol) of sodium hydride were added to the molten mPEG and
stirred at 110–120 8C until the evolution of hydrogen abated (about
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O
O
O
O
O
O
O
O
O
O
O
O
P
P
P
P
O
P
P
O
O
O
O
Et2NH, heat
O
O
TsOH, heat
Toluene, 14 h
(HCHO)n, MeOH
3
2
1
COOH
THF, 60 o
5 h
C
H2N
NH2
COOH
4
COOH
BrSi(CH3)3
hydrolysis
HN
NH
O
O
HO
OH
HN
NH
P
P
O
O
P
6
O
O
HO
HO
OH
OH
P
5
P
O
O
P
O
O
O
OH
O
P
O
OH
O
P
O
O
Figure 1. Synthesis of targeting ligand for HA binding.
NaH
100-110 oC, 24 h
O
+
Br
R
OH
n
PEG
11-bromo-undecene
7a or 7b
8
R = OCH3; 7a) or R = NHBOC; 7b)
H3CCOSH
O
R
O
n
AIBN, MeOH, Reflux
9a or 9b
PEG-Undecene
O
HCl in MeOH
Reflux, 16 h
O
R
O
n
S
10a or 10b
PEG-Undecene thioacetate ester
O
R
O
n
S
2
11a or 11b
PEG-AlkSH
R = OCH3; methoxy poly(ethylene glycol)-undecyl mercaptane (
) or
11a
)
R = NH2; amino poly(ethylene glycol)-undecyl mercaptane (
11b
Figure 2. Synthesis of the bifunctional PEG undecylmercaptane.
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20 min). 11-Bromo-1-undecene (8) (2.33 mL, 10 mmol) was added to
the reaction flask and the mixture was stirred at the same
temperature overnight to form the ether product. At the end of the
reaction, the remaining hydride was quenched by the addition of
30 mL of methanol. After evaporation of methanol, the residue was
dissolved in toluene, filtered to remove inorganic salts, which
precipitated in toluene. Finally, toluene was evaporated and the
obtained residue was dissolved in acetone and precipitated in cold
diethyl ether to yield the product methoxy PEG-undecene (9a) with
thefollowing 1H NMRspectrum[dH (CDCl3):5.8 (m, 1H, ꢁCH¼), 4.95
(t, 2H, CH2¼), 3.5–3.9 (m, ꢂ180 H, ꢁOCH2CH2ꢁ), 3.45 (m, 2 H,
ꢁOCH2ꢁCꢁCꢁC), 3,3 (s, 3 H, CH3Oꢁ), 2.0 (m, 2 H, ꢁCH2CH¼) and
1.7–1.2 (m, 14 H, the remaining protons of the alkene part)]. For
the addition of thioacetic acid to the terminal double bond, 4.4 g
(2 mmol) of the intermediate 9a and 0.480 g (3 mmol) of AIBN were
dissolved in 30 mL of dry methanol (dried by using a molecular
performance liquid chromatography (HPLC) to determine conver-
sion and purity of the synthesized compound (Figure 4, above
spectrum) [dH (CDCl3) 3.5–3.9 (m, 180 H, ꢁOCH2CH2), 3.45 (m, 2 H,
ꢁOCH2ꢁ), 3.3 (s, 3 H, CH3Oꢁ), 2.55 (tt, 2 H, ꢁCH2SH) and 1.7–1.2 (m,
18 H, the remaining protons of the alkane part)].
A similar reaction scheme (Figure 2) was applied for the
synthesis of the corresponding amine derivative of the polymer
with some slight modifications due to the present amine group.
Amine-group-protected t-butoxycarbonylamino-PEG (BOC-NH-
PEG) was used instead of mPEG in order to prevent the alkylation
of the amine group during the Williamson ether synthesis. The
corresponding PEG monoamine was synthesized by polymeriza-
tion of ethylene oxide with potassium bis(trimethylsilyl)amide as
anionic initiator according to methods described in literature.[30,31]
The terminal amine group was then protected by BOC by stirring it
overnight in a mixture of dioxane and water in the presence of
potassium hydroxide.[32] The efficiency of obtained amine protec-
tion was examined by measurement with the amine reactive
fluorescamine.[33] After amine group protection, the reaction with
11-bromundecene was completed as described above for the
methyl terminated PEG. In the final step, the used methanolic HCl
hydrolyzed not only the thioacetate ester to free thiol, but also
deprotected BOC protected amine to yield the free amino PEG
undecylmercaptane (11b), which was precipitated as described
above for 1H NMR (Figure 4, low spectrum) [dH (CDCl3) 3.5–3.9 (m,
180 H, OCH2CH2), 3.45 (m, 2 H, ꢁOCH2ꢁ), 2.8 (t,t, 2 H, ꢁCH2NH2),
˚
sieve of 4 A). 2 mL of thioacetic acid were added and the reaction
mixture was stirred under reflux for 72 h. Finally, remaining
methanol was evaporated using the rotary evaporator and the
obtained polymer residue was dissolved in acetone and subse-
quently precipitated in cold diethyl ether yielding compound
10a.[28] The following hydrolysis of the thioacetate ester 10a to the
free thiol group was carried out in methanolic HCl as described by
Bain et al.[29] to give the mPEG undecylmercaptane (11a), which
was purified by repeated precipitation in cold diethyl ether. The
synthesized polymer was characterized by 1H NMR and high-
O
N
O
OH
O
O
O
HN
NH
O
O
DCC, NHS
O
O
HN
NH
O
O
P
P
O
O
Dioxane, 2h
5
P
P
O
O
O
P
O
O
P
O
O
O
O
P
O
O
P
O
12
O
O
O
O
O
SH
H2N
O
n
5
H
H
O
SH
O
O
SH
N
O
N
O
O
n
5
n
5
HN
NH
HN
NH
O
O
O
O
O
O
HO
OH
SiBr(CH3)2 , CH2Cl2
MeOH
P
P
P
P
O
O
14
HO
HO
OH
OH
13
O
P
O
O
P
O
P
O
O
P
O
O
OH
OH
Figure 3. Synthesis of the BP conjugated polymer.
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Figure 4. H NMR spectrum of mPEG undecylmercaptane (top) and amino PEG undecylmercaptane (bottom).
2.55 (tt, 2 H, ꢁCH2SH), and 1.7–1.2 (m, 18 H, the remaining protons
of the alkane part)].
and subsequently loaded onto DEAE-sephadex A25. From there it
was eluted using 0.2 M ammonium carbonate, which was finally
dried and evaporated using a freeze dryer. To remove non-
evaporated buffer salts and non-bound BP an additional dialysis
step was added using de-ionized water and a dialysis tubing with a
of MWCO of 1 kDa.
The amine terminate polymer was finally conjugated to the BP
carrying benzoic acid using standard carbodiimide chemistry as
depicted in reaction Figure 3. Briefly, 1.5 g (2 mmol) of the still ethyl
protected BP compound 5 were dissolved in 50 mL of dry dioxane
˚
(dried by 4 A molecular sieve). To this solution, 0.46 g of NHS
(4 mmol) and 0.824 g (4 mmol) of DCC were added and the obtained
solutionwas stirredfor 2 h atroom temperature in ordertoactivate
the carboxyl group of the BP for the reaction. After that, 2.2 g
(1 mmol) of the amine terminated PEG undecylmercaptane (11b)
were added and the flask was further stirred for 2 d. Afterward, the
precipitated dicyclohexylurea was filtered off, and the solvent
dioxane was evaporated under vacuum, the obtained product was
again dissolved in acetone and precipitated twice in cold diethyl
ether, and finally collected by suction filtration and subsequently
dried under vacuum. To obtain the final product bearing the free BP
group, the ethyl groups of the esters were removed by subsequent
mild hydrolysis using bromotrimethylsilane as reagent. 3 g of the
compound 13 were dissolved in 10 mL of dichloromethane and
cooled to 0 8C. 5 mL of bromotrimethylsilane were added drop-wise
to this solution and the mixture was further stirred at room
temperature for 2 d. After evaporation of dichloromethane, 20 mL
of methanol were added and the mixture was stirred overnight
and subsequently methanol was removed by heat treatment. The
dried final product was dissolved again in acetate buffer (pH ¼ 4)
1
2.2. H NMR Spectroscopy
For NMR spectra of the products and intermediates, they were
dissolvedin CDCl3 withTMSasinternalstandardandsubsequently
measured using Bruker Avance 600 spectrometer (Bruker Biospin,
Rheinstetten, Germany).
2.3. Reverse-Phase (RP) HPLC
Samples of the synthesized polymers were investigated by HPLC
using a setup consisting of a DGU-14A degasser, LC-10-AT pump,
SIL-10 AD autosampler, and SCL-10A VP controller (Shimadzu,
Duisburg, Germany). A linear gradient of 20–100% solvent B (90%
acetonitrile in water) in solvent A (10% acetonitrile in water) over
30 min was applied as mobile phase at a flow rate of 1 mL ꢀ minꢁ1
.
50 mL samples (5 mg ꢀ mLꢁ1) were separated at 40 8C using a
˚
macroporous reverse-phase column (PLRP-S 300 A, 5 mm, Polymer
Laboratories, Amherst/MA, USA) and the corresponding PLRP-S
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guard column. The samples were subsequently detected by a
low-temperature evaporative light scattering detector (ELSD-LT
Shimadzu GmbH, Duisburg, Germany).
the citrate coating with the thiolated polymers on the particle
surfaces. Excess of unbound polymer was subsequently removed
by four times ultrafiltration at 750 g.[36]
2.4. Polymer Binding to Hydroxyapatite or Gold
2.7. Particle Size Analysis and Zeta Potential
Measurements
The dual functionalities of the polymer were explored by testing its
affinity to HA powder (due to the presence of BP groups) and gold
surfaces (due to the presence of the alkanethiol part).
The average particle size, polydispersity index, and the zeta
potential of the prepared GNPs were analyzed by photon
correlation spectroscopy using a Zetasizer 3000 HAS (Malvern,
Instruments GmbH, Germany). 300 mL of the obtained GNP
dispersions were added to 3 mL of Millipore water immediately
beforemeasuringtheirsizeandzetapotentialatroomtemperature.
The refractive index and the viscosity of water were used for
calculation of the results.
For HA binding, 2 mL of the polymer modified with free BP
(1 mg ꢀ mLꢁ1) were added to a centrifuge tube containing 10 mg
of accurately weighed HA powder. The tube was agitated in a
horizontal shaker adjusted to 200 rpm at room temperature
overnight. After that, the tube was centrifuged at 3000 rpm and
the liquid supernatant was subsequently analyzed by RP-HPLC.
Binding to gold surfaces was tested according to the following
procedure: 1 mL of free BP modified polymer solution (1mg ꢀ mLꢁ1
)
wasaddedtofourglasscoverslips(18 mmꢃ 18mm)coatedbyathin
layer of gold at room temperature. After 1 d of shaking, the solution
was analyzed for the presence of the polymer again by RP-HPLC.
2.8. Transmission Electron Microscopy (TEM) Imaging
of the Nanoparticles
10 mL of the nanoparticle samples were dropped on copper grids
(Plano Formvar/Carbon films on 400 mesh grids, 3.05 mm dia-
meter)and dried at room temperature overnight beforetakingTEM
images (Zeiss EM 10 C/CR, Germany).
2.5. Preparation of Colloidal Gold Nanoparticles
Gold nanoparticles (GNPs) were synthesized by citrate reduction of
tetrachloroaurate tri-hydrate according to literature with only
slight modifications;[34,35] citrate stabilized nanoparticles of
approximately 40 nm in diameter were obtained using the
following conditions, 0.5 mL of 1% HAuCl4 ꢀ 3H2O was added to
100 mL of Millipore water in a 250 mL round flask. The solution was
heated to reflux under vigorous stirring. 1 mL of water containing
170 mg of trisodium citrate was then added rapidly to the boiling
solution. The addition of the citrate salt resulted in gradual change
ofthesolutioncolorfrompaleyellowtopinkandfinallytodeepred.
Boiling and stirring was continued for another 20 min. After that,
the heating jacket was removed and the particle dispersion was
cooled to room temperature under stirring. The obtained colloidal
dispersionwasthencentrifugedatlowspeed(2500 rpm)for15 min
to remove coarse particles.
2.9. Dispersion Stability of GNPs
High ionic strength salt solution (5 M NaCl), BSA (200 mg ꢀ mLꢁ1) and
diluted bovine serum (10 vol%) were added to the particles to test
the dispersion stability of coated GNPs in comparison with citrate-
stabilized nanoparticles. The stability of the colloidal dispersions
was monitored by size determination, zeta potential measurement
and by changes in the UV-Vis spectra (data not shown) of
nanoparticle dispersions.[37,38]
2.10. Adsorption of Bisphosphonate-Functionalized
GNPs to Endobone
GNPs functionalized with different amounts of GNP-BP in the
coating were used to study their adsorption behavior to HA
granules (Endobone) compared to nanoparticles coated with only
mPEG undecylmercaptane as control. 3 mL of the colloidal gold
dispersions were added to 0.5 g of Endobone granules (size: 2.85–
5.6 mm) and incubated for different time intervals at room
temperature on a horizontal shaker set to 200 rpm. The percentage
of nanoparticles adsorbed to the HA was calculated from the
change of UV absorption determined at maximum absorption
wavelength of the particles to determine the amount of particle
loss,
2.6. Modification of Nanoparticle Surfaces with
Different Polymers
mPEG undecylmercaptane and the BP modified PEG polymer were
used to coat the originally citrate stabilized GNPs. Excess amounts
of the polymers were added to the nanoparticle dispersions to
ensure a complete coverage of the particle surfaces. For the
preparation of GNPs coated with mixed polymers, a parallel
adsorption method for the different polymer types was applied.
Thepolymers, mPEGundecylmercaptane(11a)and theBPmodified
polymer (14), were added to the nanoparticle dispersion (total
concentration of the polymer mixture; 5 mg ꢀ mLꢁ1 gold dispersion).
For example, 50 mL of the first polymer solution (1 mg ꢀ mLꢁ1) and
50 mL of the second polymer solution (1 mg ꢀ mLꢁ1) were mixed
together and added to 20 mL of the nanoparticle dispersion to
obtain GNPs coated with expected 50% of each polymer. The
resulting dispersion with polymers was mixed overnight in a
horizontal shaker set to 200 rpm to allow a complete exchange of
Absbefore ꢁ Absafter
%Adsorbed ¼
ꢀ100%
(1)
Absbefore
where ABSbefore is the absorbance of nanoparticles at lmax before
incubation and ABSafter is the absorbance of nanoparticles at lmax
after incubation.
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3. Results and Discussion
Radical addition of thioacetic acid to the double bond of
compound 9a was performed by applying heat in the
presence of AIBN as radical initiator. This reaction follows
anti-Markovnikov rule and yields acetate protected thiol
derivatives (10a) with a high conversion (above 95% as
estimated with NMR). The 1H NMR spectrum of compound
10a is characterized by the disappearance of the signals
attributed to the double bond of the alkene located at
d ¼ 5.8, 4.95, and 2.0 and the appearance of new signals at dH
(CDCl3) ¼ 2.85 (t, 2 H, ꢁCH2S) and 2.3 (s, 3H, ꢁSCOCH3) for
the thioacetic acid part. There were no obvious changes
observed for other proton signals, which are related to
mPEG chain and the aliphatic part of the molecule. The final
deprotection of the of the thioacetate group of compound
10a to free thiol was carried out under acidic condition
using anhydrous solution HCl in MeOH.[39,40]
3.1. Synthesis of 3,5-Di(tetraethylethylamino-2,2-
bisphosphonate)benzoic Acid
The BP ester 5 was chosen as targeting ligand, since it is
suited to overcome problems associated with the commer-
cially available BPs, like solubility in organic solvents or the
lack of possible conjugation schemes to the PEG derivatives.
The used BPs instead have the following advantages: firstly,
two BP groups are contained in one attached molecule,
which then consequently has a higher HA binding affinity
compared to compounds containing only one BP group.
Secondly, the ease of detection due to the presence of the
aromatic benzene ring, which can be detected using its
fluorescence or alternatively by UV absorption, which
is sufficient to distinguish it from the unmodified PEG
polymer. Furthermore, the compound provides a free
carboxylic acid group for subsequent easy conjugation
withprimaryaminesusingDCCchemistry. Thesynthesisof
the ester used for conjugation [3,5-di(tetraethylethyla-
mino-2,2-bisphosphonate)benzoic acid; 5] is depicted in
Figure 1. The synthesis started from the BP compound 1,
which underwent methoxy methylation with methanol
and paraformaldehyde in the presence of diethylamine to
yield the intermediate 2 (first step).
PEGmonoaminewithamolecularweightof2000Dawas
synthesized as reported earlier.[31] The actual number
molecular weight of the polymer was confirmed by 1H NMR
after modification with trifluoroacetic acid anhydride. The
results indicated that the obtained average molecular
weight of NH2-PEG-OH was about 1960 Da. For the further
reaction the primary amine of NH2-PEG-OH was protected
with an excess of BOC anhydride in presence of potassium
hydroxide solution. Under these alkaline conditions, BOC
protection is only achieved for the amine group, since
the diester formed with the hydroxyl group is again
cleaved under these reaction conditions. Based on a
standard fluorescamine assay, it was found that the
protected polymer contains less than 1% free amine
indicating a sufficient protection of the terminal amine
with BOC [the 1H NMR spectrum of BOC-NH-PEG-OH is
characterized by the following signals; dH (CDCl3) ¼ 3.5–3.9
(m, 180 H, ꢁOCH2CH2ꢁ) and 1.45 [s, 9 H, ꢁC(CH3)3].
The unstable intermediate 2 was then converted to the
unsaturated compound 3 by elimination of methanol,
catalyzed by refluxing it with trace amounts of p-
toluenesulfonic acid in toluene. Methanol is thereby
removed from the reaction mixture by calcium hydride
using a Soxhlet apparatus (step 2). The obtained chemical
shifts of both compounds 2 and 3 are in a good agreement
with that described in the literature.[26,27]
Compound
5 was obtained by anti-Markovnikov
The BOC-protected PEG monoamine was then reacted
with 11-bromo-1-undecene in the presence of sodium
hydride as described for the methoxy derivative to yield the
protected PEG monoamine undecene as described in
Figure 2. The use of the BOC-protected polymer prevented
the possible alkylationof the aminegroup to take place. The
reaction was then completed with the thiol addition and
the hydrolysis of the protective groups as described for
the synthesis of methoxy PEG undecylmercaptane. [The
1H NMR spectrum of pure amino PEG undecylmercaptane
(11b) is presented in Figure 4 (bottom)].
addition of the amine groups of 3,5-diaminobenzoic acid,
compound 4, to the double bond of intermediate 3
(step 3).
3.2. Synthesis of the PEG Undecylmercaptane
Derivatives
Alkanethiol terminated PEG monomethyl ether was
obtained through a three-step synthesis as described in
Figure 2. For the reaction the PEG monomethyl ether (7a)
was deprotonated by 10 equiv. of sodium hydride and
subsequently reacted with an excess of the 11-bromo-1-
undecene (8) in order to obtain a complete modification of
the added PEG polymers to mPEG undecene (9a). Subse-
quentlythepolymerswereprecipitatedincoldether, which
is a good solvent for eventually residual alkene. The
terminal double bond of the alkene part is not reactive
under these conditions and therefore did not need any
protection.
3.3. RP-HPLC
HPLC chromatograms of the different synthesized and used
polymers are presented in Figure 5, the chromatogram of
the mPEG undecylmercaptane [mPEG-AlkSH] is character-
ized by two neighboring peaks. The first peak eluted at
19.5 min is related to the reduced (singly present) portion of
the synthesized polymer and the second peak eluted at
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polymer. The reaction was carried out
applying standard DCC and NHS chem-
istryinnon-aqueousmediaasdepictedin
Figure 3. The molar ratio between the BP
and the polymer was kept at 2:1, which is
low compared to the ratios used in
literature, to avoid a modification of both
ends of the polymers with BP (thiol
should be kept free for the binding to
gold).[42] Themeasured1HNMRspectrum
of the product showed the characteristic
peaks of both parts of the polymer and of
the BP. Bromotrimethylsilane supported
hydrolysis of the ester group of the BP
resulted in the free BP modified
polymer (14).
The estimated BP content of the poly-
mer as determined from a phosphate
Figure 5. HPLC chromatograms of different undecylmercaptane derivatives and the raw
materials: mPEG, mPEG undecylmercaptane (mPEG-AlkSH) and after oxidation with air, assay[43] was 97 ꢄ 2.32% of the theore-
PEG monoamine (H2N-PEG) and amino PEG undecylmercaptane (H2N-PEG-AlkSH).
tical value, when calculated as inorganic
phosphorus (using ibandronate for the
standard curve) after oxidation and
21.1 min is related to the oxidized, dimeric portion of the
polymer. Upon complete oxidation with pressurized air all
polymer species are dimerized, resulting in a single peak at
21.8 min. Both of alkylated polymer species are retained
longer on the solvent material, which is related to the
increased hydrophobicity compared to methoxy PEG
(15.3 min). The oxidized polymers are generally eluted at
the later time point, because the oxidized form has a higher
molecular weight and at the same time it is less hydrophilic
due to the presence of the disulfide bond.
complexation with molybdate reagent. This indicates a
successful binding of the phosphate to the polymer and a
successful removal of excess of BP by the dialysis.
3.4. Binding to Hydroxyapatite and Gold Surface
The final BP modified polymer (14) exhibited a high affinity
to gold and HA, which was determined by incubation with
both substances. HPLC chromatography showed the
complete disappearance of the characteristic polymer
peaks after incubation with HA or gold (data not shown).
Polymers without the respective functional groups did not
disappear from the solution. The high effectiveness of the
removal of the polymer by both gold and HA confirmed the
presence of both functional groups, the free BP groups and
the presence of thiol group.
The chromatogram of the corresponding PEG mono-
amine is also characterized by a single peak eluted at
14.6 min, which is significantly broader than the methoxy
derivative since it is obtained by custom synthesis and
furthermore could be also be differently charged due to a
protonation of the amine group.[41] In case of the modified
amino PEG undecylmercaptane [NH2-PEG-AlkSH], also two
peaks can be detected in the chromatogram. One peak
eluting at 17.9 min due to the reduced portion and the other
peak eluted at about 19.6 min, which is due to the oxidized
portion of the polymer. The much broader peaks obtained
for these polymers are due to the wider molecular weight
distribution of the PEG monoamine and the above
mentioned additional widening due to possible charges
on the amine function. The polymer derivatives carrying
the BP could not be compared using the same chromato-
graphic setup, since we had to add allylamine to the eluent
in order to compensate the acid functions.
3.5. Preparation of Colloidal Gold Nanoparticles
GNPs
were
prepared
by
the
reduction
of
HAuCl4 ꢀ 3H2O solution using trisodium citrate as reducing
agent. The prepared GNPs were obtained with ruby red
color and proved to have the characteristic surface plasmon
resonance at 524 nm, corresponding to their size of
40 nm.[44] Upon coating with the polymers this resonance
peak also exhibited the characteristic red shift of 8 nm,
indicating successful modification of the particle sur-
faces.[45–47]
For the final modification of the amino PEG undecyl-
mercaptane the unhydrolyzed BP ester was linked to the
polymer via an amide bond between the carboxylic acid
group of the BP and the primary amine terminus of the
The sizes of different preparations of GNPs were
determined by photon correlation spectroscopy (Table 1).
Different citrate stabilized GNP preparations range from
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Table 1. Sizes, polydispersity indices (PI), and zeta potentials of different batches of citrate stabilized and polymers coated GNPs determined
by photon correlation spectroscopy.
GNP preparation
Hydrodynamic
Polydispersity index
Zeta potential [mV]
size by PCS [nm]
citrate-coated GNPs (batch 1)
citrate-coated GNPs (batch 2)
citrate-coated GNPs (batch 3)
citrate.coated GNPs (batch 4)
batch 1 coated with mPEG-AlkSH
batch 2 coated with NH2PEG-AlkSH
batch 3 coated with BP-PEG-AlkSH
46.1 ꢄ 1.12
44.2 ꢄ 0.436
38.6 ꢄ 0.153
41.8 ꢄ 1.03
57.3 ꢄ 3.11
55.5 ꢄ 1.39
45.2 ꢄ 1.1
0.479 ꢄ 0.068
0.543 ꢄ 0.055
0.416 ꢄ 0.08
0.395 ꢄ 0.018
0.426 ꢄ 0.061
0.578 ꢄ 0.005
0.465 ꢄ 0.014
ꢁ48.7 ꢄ 5.1
ꢁ46.0 ꢄ 4.5
ꢁ47.7 ꢄ 3.2
ꢁ51.9 ꢄ 8.9
ꢁ19.4 ꢄ 6.1
ꢁ02.1 ꢄ 5.3
ꢁ44.6 ꢄ 7.1
38.6 ꢄ 0.153 to 46.1 ꢄ 1.12 nm with polydispersity indices
around 0.5. Modification of the nanoparticles with thiol
containing polymers slightly increased their hydro-
dynamic size as expected due to formation of a PEG
layer around the nanoparticles. The measured size increase
of the GNPs ranged from 7 to 12 nm, corresponding to
polymer layers of 3.5 to 6 nm, which agrees well with
literature.[38,48]
The surface charges of different coated GNP preparations
are also listed in Table 1. The zeta potential measurements
show that the surface charge of bare (citrate stabilized)
GNPs is negative with approximately ꢁ48.5 ꢄ 5.5 mV,
which is attributed to the adsorption of the negative citrate
anions. Appropriate particle stability is obtained with zeta
potentialsjlargerthan30 mV,duetoelectrostaticrepulsion
forces between similarly charged particles, which compen-
sates attractive van der Waal forces.[49,50]
To further characterize the morphology and to verify the
size of the prepared nanoparticles, imaging by transition
electron microscopy was applied. The obtained citrate
stabilized GNPsarespherical, all smallerthan about100 nm
and well-separated from each other when dried on the
carbon coated copper grid (Figure 6A). Upon examination of
the nanoparticles with higher magnifications it was found
that most of the synthesized nanoparticles are spherical
withonly fewofthemexhibitinga slightlypolygonal shape
(Figure 6B). The polymer coated particles did not exhibit
a different morphology due to the weak contrast of the
polymer shell.
Coating of citrate stabilized GNPs with mPEG-AlkSH
resulted in an increase of the zeta potential from about
ꢁ48.7 ꢄ 5.1 to about ꢁ19.4 ꢄ 6.1 due to the substitution of
some the citrate anions adsorbed on the particle surface by
the thiol groups of the polymer.[51] The PEG-coated GNPs
still remained well dispersed, but instead of the electro-
static repulsion due to the steric repulsion of the polymer
chains attached to the particle surfaces.[52] For GNPs
carrying the BP ligand a highly negative charged particle
surface was observed with zeta potentials similar to that of
the citrate stabilizedGNPs (Table 1), whichcan beexplained
Figure 6. TEM images of the prepared citrate stabilized GNPs; overview (12.500 ꢃ) (A) and higher magnification (80.000ꢃ) (B).
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with the presence of additional anionic BP groups on the
particle surface.[53,54]
interactions of cysteine residues of the supplied proteins
and GNPs or an unspecific adsorption of proteins on the
nanoparticles, which both results in a shielding of the
negative charges of the GNPs and subsequent aggregation.
In contrast, the polymer-protected GNPs are either not
affected at all (in case of the mPEG-AlkSH coated GNPs) or
show only a slight size increase (in case of BP-PEG-AlkSH
coated GNPs) by addition of BSA and serum. The protein
resistance (i.e., the stability) of PEG-coated GNPs is a
consequence of the high stability of the interfacial water
layer, which prevents direct contact between the particle
surface and the proteins.[24] The slight increase in particle
sizeafteradditionofBSAandserumtonanoparticles coated
with BP-PEG-AlkSH (Figure 7) can be attributed to the
presence of negative charges of the BP groups, which might
interact with positively charged groups of the proteins.
3.6. Dispersion Stability of GNPs
For the intended biological applications of the GNPs, the
stabilityofthedifferentpreparationswasinvestigatedinthe
presence of high ionic strength media and in the presence of
blood proteins (BSA and bovine serum). The addition of 5 M
NaCl to bare GNPs resulted in a rapid aggregation of the
nanoparticle monitored bydrasticchange oftheir color from
rubyred tovioletandfinally toa colorless solution due to the
subsequentprecipitationofthenanoparticleaggregates.The
characteristic surface plasmon resonance disappeared and a
newbroadpeakatmuchhigherwavelengths(above600 nm)
was observed due to the formation of larger gold aggregates.
The instability of citrate coated GNPs in the presence of high
concentrations of NaCl could also be monitored by a large
increase of the hydrodynamic size as measured by photon
correlation spectroscopy (Figure 7). The GNP aggregation
induced by addition of NaCl is due to the reduction of the
electrostatic repulsion between surface-charged nanoparti-
cles caused by the added sodium and chloride ions and
consequently the decreasing distance between the particles
favoring the interaction via van der Waals forces.[55,56] Both
polymer-coated GNPs on the other hand are not affected by
NaCl, as showninFigure7, because theyare notstabilizedby
the surface charge but by the steric repulsion effect of the
polymer chains.[37]
3.7. Adsorption of Gold Nanoparticles to
Hydroxyapatite
The adsorption of BP modified GNPs (GNPs-BP) to HA was
studied to evaluate the binding of ligand-modified GNPs to
bone mineral, intended as target tissue. For this study
commercial Endobone granules were used as a porous HA
ceramic matrix, which is produced by sintering of bovine
bone and consequently possess similar structure as natural
bone. The porous template is lacking all tissues, cell and
proteins, which are destroyed during the sintering step.[57]
The intensity of HA binding was evaluated using GNPs
modified with different amounts of BP in the polymer layer
and was compared to the binding of GNPs coated with
mPEG-AlkSH alone as control (0% BP). The UV measure-
ments of the decanted GNP particle solutions showed that
increasing the amounts of ligand (BP) led to more GNPs
adsorbed to the HA granules as depicted in Figure 8.
Similarly to the sodium chloride addition, citrate-coated
GNPs rapidly aggregated after addition of BSA as well as
native blood serum, which could be demonstrated by the
disappearance of the characteristic surface plasmon
resonance (data not shown) and by their large size increase
measured by photon correlation spectroscopy (Figure 7).
These observations could be attributed to the specific
Control GNPs showed no measurable binding to HA,
while about 25% of the initially added GNPs modified with
25% BP (75% mPEG-AlkSH) already adsorbed partially to HA
within the first 24 h. The adsorbed amount increased to 40%
after 48 h with no further increase in the adsorbed amount
despite further agitation of the particles suspension (up to
4 d). The bound amounts of particles modified with 50% BP
were about 45% after 1 d and reached 64% after 2 d. The
adsorbed amounts of GNPs modified with 75 and 100% BP
were 58 and 37% after 1 d, and 76 and 81% after 2 d and
reached 86 and 100% after 4 d, respectively. The steady
increase in the adsorbed amounts with increasing con-
centration of BP in the particle surface can be explained by
the higherHAbinding strength of GNPs containing moreBP
in their surface. The relatively long time (about two days) to
establish the equilibrium between the adsorbed and free
particles can be attributed to the necessary diffusion of the
particles within the large volume of the dispersion medium
(3 mL) and the necessary penetration into the macro and
Figure 7. Size of differently modified GNPs (& citrate-coated,
PEG-AlkSH coated and & BP-PEG-AlkSH-coated) and the effect of
addition of NaCl, BSA and 10% bovine serum on the particle size.
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Figure 9. HA binding of BP-modified GNPs in the absence and
presence of calcium chloride.
Figure 8. HA binding of GNPs modified with different amounts of
BP (e.g., 75% means that GNPs are coated with 75% BP-PEG-AlkSH
and 25% mPEG-AlkSH).
binding of the synthesized polymers to HA as well as gold
surfaces. The synthesized polymers possess a high affinity
for gold surfaces due to the functionalization of the
hydrophilic PEG polymer by a terminal thiol containing
alkylgroup, whichfurtherprovidesanincreasedstabilityof
the applied surface coatings. The additionally added BP
moiety, is known for its specific affinity to HA contained in
bone tissue and therefore if highly promising for bone
targeting applications.
micro pores of the small endobone granules on the bottom
of the flasks to reach their adsorption sites. Similar results
are obtained by many other authors who studied the
binding of nanoparticles or liposomes to HA powder.[53,58]
Since it is well known that BP strongly binds to divalent
ions, such as calcium,[59] the binding assay of BP-modified
GNPswasrepeatedalsoinpresenceofpotentiallyinterfering
soluble calcium chloride to explore the effect of calcium ions
on the affinity of the nanoparticles. The used calcium
concentration was 2.5 ꢃ 10ꢁ3 M, which is the physiological
concentrationpresentinbloodserum.Thebindingaffinityof
100%BP-modifiedGNPstoHAinthepresenceandabsenceof
calcium chloride is depicted in Figure 9. It could be observed
that there is no difference in the binding of nanoparticles
either in presence or absence of calcium. This indicates that
the binding affinity of particles to HA is not affected by the
presence of calcium ions in the colloidal solution, which is
essential for a later application in vivo.
The synthesized polymers further more were applicable
for the modification of GNPs prepared by citrate reduction.
The polymer coated particles were stable under different
conditions commonly aggregating unprotected particles,
such as high ionic strength NaCl solutions as well as
solutions containing BSA and serum. The prepared polymer
modified GNPs can therefore be considered sufficiently
stable under in vivo conditions.
The accordingly modified particles demonstrated a high
affinity to bone mineral, which could be adjusted by
altering the amount of BP targeting ligand in the particle
coating. The particles affinity was neither diminished by
physiologic calcium concentrations in the solution nor by
proteins or serum present in the particle suspension.
Furthermore, the in vitro binding of BP-modified GNPs
was carried out in presence of BSA (200 mg ꢀ mLꢁ1) and
serum (10 vol% to determine, if the already observed
interaction of BP with these proteins compromises the
binding to HA granules. The results of these experiments
are presented in Figure 10, which shows that the binding
affinity of nanoparticles to HA is not affected by the
presence of BSA or serum. The obtained data indicate that
the BP-modified GNPs are stable and the observed
interaction of particles and proteins, especially BSA
(Figure 7), does not influence their bone mineral affinity.
4. Conclusion
Figure 10. HA binding of BP-modified GNPs in the presence of BSA
(200 mg ꢀ mLꢁ1) and bovine serum (10 vol% compared to an aqu-
eous dispersion.
Bifunctional thioalkylated PEG derivatives were success-
fully synthesized, which was proven by the efficient
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Based on the conducted in vitro studies, the BP-
functionalized GNPs seem to be a promising model for
the investigation of the particle-size-dependent in vivo
bone targeting of nanoparticles. An adjustment of the
binding strength can be achieved by changing the amounts
of immobilized BP-modified polymers. An exchange for
other suitable ligands would furthermore allow using the
here proposed PEG coated nanoparticle system also for
other targets and the exploration of other applications than
bone.
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online: DOI: 10.1002/mabi.201200046
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