Gold nanoparticle-cored poly(propyleneimine) dendrimers as a new platform for multifunctional drug delivery systems

Article abstract of DOI:10.1039/c1nj20206e

Two poly(propyleneimine) dendrons (PPI dendrons) of third generation, containing eight branches (nitriles or primary amines) and having tyramine as the focal point, were prepared. A tetraethylene glycol spacer (TEG), terminated by a protected thiol group,

Full text of DOI:10.1039/c1nj20206e

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PAPER
Gold nanoparticle-cored poly(propyleneimine) dendrimers as a new
platform for multifunctional drug delivery systemswz
Marie-Christine Daniel,*a Margaret E. Grow,a Hongmu Pan,a Maria Bednarek,b
William E. Ghann,a Kara Zabetakisa and Joseph Cornishc
Received (in Gainesville, FL, USA) 5th March 2011, Accepted 31st July 2011
DOI: 10.1039/c1nj20206e
Two poly(propyleneimine) dendrons (PPI dendrons) of third generation, containing eight
branches (nitriles or primary amines) and having tyramine as the focal point, were prepared.
A tetraethylene glycol spacer (TEG), terminated by a protected thiol group, was attached to their
focal points. The obtained TEG–PPI dendrons were successfully used to cap 18 nm spherical gold
nanoparticles, with no need for intermediate thioctic acid exchange of citrate, as previously
reported for non-anionic ligands. The novel gold nanoparticle-cored PPI dendrimers were
characterized by UV-visible and FT-IR spectroscopic techniques, dynamic light scattering (DLS),
zeta-potential and transmission electron microscopy (TEM). Importantly, the amine terminated
gold nanoparticle-cored PPI dendrimer showed great stability in PBS buffer, as well as in the
presence of a salt, and they did not show any sign of degradation after freeze-drying. Also, this
new type of PPI dendrimer was found to protonate at pH around 4.5 for their internal tertiary
amines. The latter being present in large numbers on each gold nanoparticle, their protonation at
pH below 5 will produce a proton-sponge effect, which plays a crucial role in lysosomal escape
during drug delivery.
displaying more than 100 000 branches,10 this requires many
steps and defects at the branches’ termini appear when reaching
Introduction
During the past decades, dendrimers have been increasingly
utilized for biological applications.1–5 Because of their nano-
in order to create large multifunctional dendrimers.
very high generations. There is thus the need for new strategies
scale size and their branched structure, they allow a longer
Dendrons differ from dendrimers in their structure by the
circulation time in the body and they can carry a larger
presence of a focal point that is functionalized differently than
the periphery.11 This focal point allows dendrons to be grafted
payload. Poly(amidoamines) (PAMAM) and poly-
onto other molecules or surfaces.12 This is of particular interest
(propyleneimine) (PPI) dendrimers are now commercial and
they have already demonstrated true potential for biomedical
applications.6–9 Although the polyvalence of dendrimers
the ability of being easily incorporated in the preparation of
for biomedical applications because it provides dendrons with
represents a great asset, one drawback is the fact that they
multifunctional systems. In fact, optimal biomedical devices
usually display a single type of termini, and their multi-
often require several different properties that are very challenging
functionalization remains a challenge. Moreover, even though
(or impossible) to find simultaneously on the same molecule, so
it was shown possible to construct giant dendrimers,
that different entities need to be combined to fulfill the intended
effect(s). Dendrons can be of great use in this matter since they
a Department of Chemistry and Biochemistry, University of Maryland
Baltimore County, Baltimore, MD, USA.
can be combined with either other dendrons, a variety of
dendritic cores, polymers or surfaces.12
E-mail: mdaniel@umbx.edu; Fax: +1 410 455 2608;
Nanoparticles have also shown great promise as nanocarriers
Tel: +1 410 455 5601
for medical purposes.13,14 Nanoparticles are good candidates as
multifunctional drug delivery systems since they present better
capabilities for hosting different functions and they provide
greater payload for each function.13–19 However, a good stability
in biological media as well as the capability of efficient lysosomal
escape is required for optimal delivery of therapeutics.20 Gold
nanoparticles in particular have been approved by FDA and, for
instance, their applications for cancer detection or treatment
have been expanding very rapidly these past years.21–25
b University of Maryland School of Pharmacy, Baltimore, MD, USA.
E-mail: mbednarek@umaryland.edu
c Department of Biological Sciences, University of Maryland
Baltimore County, Baltimore, MD, USA. E-mail: jc12@umbc.edu
w Dedicated to Prof. Didier Astruc on the occasion of his 65th birthday.
z Electronic supplementary information (ESI) available: The syntheses of
compounds 1 and 2, the 1H NMR spectra of dendrons 7, 8 and 9, TEM
pictures of GNPG3NH2, the DLS measurements of GNPG3NH2 in
1ꢀ PBS buffer, UV-vis spectra of GNPG3NH2 in the presence of a salt
and DLS measurements of GNPG3NH2 after freeze-drying are available
free of charge. See DOI: 10.1039/c1nj20206e
c
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Nanoparticle-cored dendrimers consist of a nanoparticle as
the dendritic core, surrounded by dendrons.26–34 Greater
stability has been observed with these new types of nano-
particles.35 However, to date very few nanoparticle-cored
dendrimers have been developed for medical purposes. We
report here the design, synthesis and characterization of a
novel thiolated PPI dendron, displaying eight branches, and its
use for the preparation of stable gold nanoparticle-cored
dendrimers. Since PPI dendrimers are known to protonate in
an ‘‘onion-like’’ fashion (where the odd shells protonate at
high pH first, and the even shells at lower pH),36,37 the new
nanoparticle-cored PPI dendrimers are expected to act as a
‘‘proton sponge’’ after cell entry by endocytosis and thus
induce lysosomal escape.
preparing the monotosylate intermediate 1, then adding thiourea
through nucleophilic substitution and hydrolyzing the thiourea
group into thiol.57 Mercaptotetraethylene glycol 2 has the
tendency to dimerize after a few days,57 so the thiol function
was protected with a xanthene group.58,59 The protected thiol
TEG 3 was obtained with a maximum yield of 52%, but it has
the advantage of being stable under most basic conditions
while providing convenient complete deprotection under very
specific conditions.58 For attachment to the phenol group of the
dendron, the spacer was functionalized with a bromide leaving
group. Thus, compound 3 was efficiently tosylated and then
brominated, with no loss of thiol protection, evidenced by
respective yields of 84% and 90%. The TEG spacer 5 was then
coupled to the PPI dendron of third generation (G3CN, 6) and
provided dendron 7 in quantitative yield. Sodium hydride was
used instead of the usual potassium carbonate in order to
deprotonate the phenol group of G3CN, due to the observed
instability of the xanthene group in the presence of potassium
carbonate. The coupling of the TEG to the dendron was clearly
Results and discussion
Synthesis of the thiolated PPI dendron
The syntheses of different types of dendrons have been
reported for their use in various applications.38–43 Some
dendrons are now available commercially, such as PAMAM
dendrons,44–46 ‘‘Frechet-type’’ dendrons47–49 and ‘‘Newkome-
type’’ dendrons.50–52 Concerning PPI dendrons, the dendrons
reported display either benzyl,53polystyrene54 or carboxylate34
focal points. In this report, tyramine was selected as the focal
point of the prepared PPI dendron so that the phenol group
could be easily attached to a thiolated spacer molecule. The
presence of the thiol function will facilitate the anchoring
of this PPI dendron to various entities. The synthesis of the
PPI dendron up to generation 3 (containing 8 branches) is
described elsewhere.55 Tetraethylene glycol (TEG) was chosen
as the spacer between PPI dendrons and gold nanoparticles for
two reasons: (i) to decrease the bulkiness of the dendron near the
surface of the nanoparticle, thus facilitating the anchoring onto
the nanoparticle surface and promoting higher density of the
dendron per nanoparticle (by decreasing the steric hindrance at
the gold surface); (ii) it has been shown that the incorporation of
oligoethylenes can decrease RES clearance, thus improving the
blood circulation time.56 Scheme 1 describes the synthesis of the
TEG spacer and its coupling to a PPI dendron. TEG was first
monothiolated using the isothiouronium salt method, that is by
1
evidenced by the appearance of a peak at 4.02 ppm on the H
NMR spectrum (see ESIw, Fig. S1). To our knowledge, this is
the first synthesis of a PPI dendron enabling grafting through its
focal point not only onto the metal nanoparticles’ surface but
also to other dendrons, dendrimers or macromolecules.
Preparation of the gold nanoparticle-cored PPI dendrimers
Scheme 2 represents the synthesis of the thiolated PPI dendrons
8 and 9, respectively, displaying eight nitrile branches and
Scheme 1 Reagents and conditions: (i) TsCl, NaOH, THF, 0 1C, 2 h;
(ii) thiourea, EtOH, reflux, 24 h, inert atm.; (iii) NaOH, reflux, 3 h,
inert atm.; (iv) 9-hydroxyxanthene, CH2Cl2/TFA (96/4), rt, 5 h, inert
atm.; (v) TsCl, NaOH, THF, rt, 18 h; (vi) LiBr, acetone, reflux, 24 h;
(vii) NaH (60% in oil), anhydrous THF, rt, 72 h, inert atm.
Scheme 2 Preparation of gold nanoparticle-cored PPI dendrimers
GNPG3CN 10 and GNPG3NH2 11. (i) Et3SiH/TFA/CH2Cl2
(1/2/97), rt, 18 h; (ii) BH3ꢁSMe2, dry THF, rt, 24 h; (iii) Citrate
GNP, pH 4.5, rt, 24 h.
c
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eight primary amines, as well as their use for the modification
of gold nanoparticles.
exchange on citrate GNP with neutral or cationic thiols led to
destabilization of the particles and aggregation, and that
intermediate exchange using thioctic acid could overcome this
problem.62 Although dendrons 8 and 9 are, respectively,
neutral and positively charged in water, the direct exchange
reaction on the citrate GNP was successful, leading to no
degradation or change of color of the gold nanoparticles
solution (Fig. 1 and 4). This finding could be explained by a
dendritic effect endowing the thiolated dendrons with better
steric protection ability of the gold core than a linear counter-
part ligand. Indeed, it has already been reported increased
stability of particles when the latter are coated by dendritic
ligands versus linear molecules.35,63
Thiol preparation
To obtain dendron 8, the xanthene group was quantitatively
removed by using 2% TFA in the presence of 1% Et3SiH.59
Contrary to mercaptoethyleneglycol, dendron 8 was found to
remain stable in air for at least a week (followed by mass
spectrometry and Ellman’s test). Furthermore, while dendron 7
was not soluble in water, dendron 8 was observed to be soluble
in slightly acidic water (pH around 4.5). The 1H NMR
spectrum in D2O shows the disappearance of the xanthene
group peaks, but also a downfield shift of the dendritic peaks
was observed (see ESIw, Fig. S2). Dendron 9 was formed
in excellent yield (95%) by reduction of the nitrile groups using
a large excess of the borane sulfide complex. The presence
of the latter simultaneously induced the deprotection of
the thiol group, as observed on the 1H NMR spectrum in
D2O (see ESIw, Fig. S3).
GNPG3CN (10) characterization
The nitrile terminated gold nanoparticle-cored PPI dendrimer
(GNPG3CN) was prepared through ligand exchange of the
citrate molecules by the thiolated dendron HSTEGG3CN 8.
As shown in Fig. 1A, only a slight shift of 4 nm was observed
between the surface plasmon resonance (SPR) of the citrate
GNP (519 nm) and of the GNPG3CN (523 nm). This shift is
the evidence of the strong adsorption of the thiol groups on
the gold nanoparticle surface. Also, as seen in the DLS data in
Fig. 1B, GNPG3CN 10 was obtained with a narrow distri-
bution (PDI = 0.176). The hydrodynamic diameter of the
purified gold nanoparticles increased by 6 nm in GNPG3CN
as compared to citrate GNP, indicating a thickness for
dendron 8 of about 3 nm in solution. The coating is also
observed on uranyl acetate stained TEM images (Fig. 1E), and
a thickness of 1.2 ꢂ 0.2 nm is measured under vacuum
conditions. The size of the GNPG3CN gold core measured
from TEM images (Fig. 1C and D) is 17.6 ꢂ 1.8 nm in
diameter (n = 150). Fig. 2 compares the ATR-FTIR spectra
of GNPG3CN and HSTEGG3CN. The general fingerprint
bands of dendron 8 are conserved in the GNPG3CN spectrum,
which confirms the presence of 8 on the gold nanoparticles’
surface. Because of the tripartite composition of the dendron
(TEG, tyramine and PPI structures), a precise assignment of
the absorption peaks is challenging. A tentative assignment for
the FTIR spectrum of HSTEGG3CN can be as follows: the
peaks at 2927 and 2864 cmꢃ1 are related to the asymmetric and
symmetric –CH2– stretching vibrations, respectively; the S–H
stretching band appears at 2505 cmꢃ1; the nitrile stretching
band is located at 2248 cmꢃ1; the band at around 1600 cmꢃ1 is
attributed to the CQC stretching of the aromatic ring and
maybe some hydration of the TEG part of the dendron;64 the
absorption band at around 1190 cmꢃ1 is assigned to C–N–C
stretching vibrations from PPI;65 the peak at 1126 cmꢃ1
corresponds to the C–O–C stretching vibrations of TEG;
the two bands at 798 and 829 cmꢃ1 are attributed to the
C–H bending mode from the tyramine part; and the C–S
stretching band appears at 719 cmꢃ1. As shown in Fig. 2, few
changes are present in the FTIR spectrum of 10: (i) the
disappearance of the S–H stretching band commonly observed
after adsorption on GNPs; (ii) the disappearance of the C–S
stretching peak; (iii) a large decrease of the intensities of
the bands at around 1600 cmꢃ1, at 1190 cmꢃ1 and at 798
and 829 cmꢃ1. These differences, as well as a very slight overall
Gold nanoparticles formation and ligand exchange
Citrate gold nanoparticles (citrate GNP) were prepared
following a modified Fren’s method,60 using large excess of
sodium citrate in order to improve the monodispersity of the
gold nanoparticles formed. The use of a 0.3 mM gold chloride
solution, 5% trisodium citrate solution and an ncit/nAu ratio of 8
led to the formation of gold nanoparticles with a diameter of
around 18 nm (see the TEM histogram, Fig. 1) and an
excellent polydispersity index (PDI) of 0.03 found by DLS
measurements. In order to avoid interference of the large
excess of citrate around the particles, the citrate GNP solution
was centrifuged once and resuspended in pure water before
the addition of the dendron-thiols 8 or 9. The latter were used
with an excess of 175 equiv. per theoretical ligand on the
particles (assuming that each thiol occupies 0.2 nm2 on the
surface of the nanoparticle).61 It has been reported that ligand
Fig. 1 Characterization of GNPG3CN. (A) UV-visible spectra of
citrate GNP (solid line) and GNPG3CN (dashed-dotted line); (B) DLS
measurements by intensity of citrate GNP (solid line) and GNPG3CN
(dashed-dotted line) in H2O; (C) TEM image of GNPG3CN;
(D) histogram plot of the sizes of the gold cores in GNPG3CN
(measured from 150 particles on the TEM images); (E) Stained
TEM images of GNPG3CN showing the -STEGG3CN coating.
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20, 30 and 45 mV, which could suggest partial protonation of
the tertiary amines of GNPG3CN. At pH 8, GNPG3CN is
neutral as demonstrated by its zeta potential around zero and
as expected. It was further noticed that, at pH 8, the color of
the gold nanoparticles solution changed to blue and flocculation
followed. This behavior is reversible when acidifying back the
solution, thus showing that its tertiary amines need at least
partial protonation in order to endow GNPG3CN with water
solubility. Such solubility pattern has been previously reported
for particles coated with charged ligands.68 The amines proto-
nation at around 4.5 for GNPG3CN is lower than the
reported pKa (B6.5) for the protonation of internal amines
in PPI dendrimers.37 This decrease in basicity was reported
earlier by Whitesides and coworkers for monolayer films after
adsorption on the gold surface.69 It has also been recently
observed on glycine–cysteamine protected gold nanoparticles:
the pKa for the ammonium group of glycine dropped from
9.6 to 5.5, also inducing aggregation of the particles at pH
values above the new pKa value (5.5).70
Fig. 2 ATR-FTIR spectra of dendron
8 (dashed line) and
GNPG3CN (solid line).
shift of the peaks (few cmꢃ1), have also been observed by
Shi et al.66 on 5 nm-GNPs stabilized with bifunctional thiols.
They can reflect a change in conformation of the dendron
upon adsorption onto the surface of the gold nanoparticles.67
GNPG3NH2 (11) characterization
The amine terminated gold nanoparticle-cored PPI dendrimer
(GNPG3NH2) was prepared through ligand exchange of the
citrate molecules by the thiolated dendron HSTEGG3NH2 9.
Similarly to the GNPG3CN case, a small red shift (7 nm) was
observed between the SPR band of the citrate GNP (519 nm)
and of the GNPG3NH2 (526 nm), as seen in Fig. 4. Also, DLS
data in Fig. 4 indicate a narrow distribution (PDI = 0.144) for
GNPG3NH2. From the UV-vis spectrum, the DLS data and
the TEM (vide infra), no aggregation was detected, which
differs from the observation of Shi et al. for amine terminated
Protonation behavior of GNPG3CN
The difference of surface charge between citrate GNP and
GNPG3CN is another evidence of the modification of the gold
nanoparticles by dendron 8. Indeed, as observed in Fig. 3A,
the zeta potential of the initial citrate GNP is negative, due to
the carboxylate groups of citrate, while the zeta potential of
purified GNPG3CN is neutral or positive depending on pH.
Because of the PPI structure of dendron 8, it confers
its protonation pattern37 to GNPG3CN. Fig. 3B shows the
surface charge of GNPG3CN at three different pH values. At
pH 3, its zeta potential reveals a single peak at around 45 mV,
indicating protonation of all the tertiary amines of dendron 8.
At pH 4.5, the zeta potential of GNPG3CN presents a very
wide distribution from 15 to 50 mV, with several maxima at
Fig. 3 Surface charge measurements. (A) Zeta-potentials of citrate
GNP (solid line) and GNPG3CN at pH 8; (B) zeta-potentials of
GNPG3CN at pH 3 (dashed line), pH 4.5 (solid line) and pH 8
(dashed-dotted line).
Fig. 4 Top: UV-visible spectra of citrate GNP (solid line) and
GNPG3NH2 (dashed line); bottom: DLS measurements (size by
intensity) of citrate GNP and GNPG3NH2 (solid and dashed lines,
respectively).
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Fig. 6 Stability in PBS buffer, pH 7.4. (A) UV-visible spectra
of GNPG3CN in H2O (dashed line) and in PBS buffer (solid line);
(B) UV-visible spectra of GNPG3NH2 in H2O (dashed line) and in
PBS buffer (solid line).
Fig. 5 Surface charge measurements. Evolution of zeta-potential of
GNPG3NH2 at different pH values (3, 4.5, 7.4 and 14).
ligands on GNPs.66 This difference could be imparted to the
intra-particles H-bonds between primary amines being favored
over interparticles H-bonds, due to a dendritic effect.27,52 The
hydrodynamic diameter of the purified gold nanoparticles
increased by 10 nm in GNPG3NH2 as compared to citrate
GNP, suggesting a thickness for dendron 9 of about 5 nm in
solution. This larger thickness as compared to the one found
for dendron 8 could be explained by the repulsion between
protonated primary amines, inducing stretching of the
dendron configuration.36 TEM images of GNPG3NH2 were
also recorded (see Fig. S4, ESIw) and the thickness found for
the shell was 1.2 ꢂ 0.2 nm, which is the same as in GNPG3CN
(vide supra). This is expected, because the repulsion between
protonated primary amines can no longer exist under the
vacuum conditions of TEM. The evolution of the surface
charge of GNPG3NH2 was also studied, by following its zeta
potential at different pH values. The pH values tested were 3,
4.5, 7.4 and 14. GNPG3NH3 remained soluble at all pH
values, always remaining red colored. As expected, the zeta
potential was found positive at pH values 3, 4.5 and 7.4
(though less positive as pH increased), but the surface charge
was found slightly negative at pH 14 (Fig. 5). A neutral surface
was expected at very basic pH, but this very slight negative
charge has also been reported very recently by Susumu et al.
on gold nanoparticles coated with amine-containing ligands.71
This phenomenon could be explained by the presence of some
hydroxide ions trapped in between the branches of the dendrons.
The slight negative charge at basic pH may be the reason for
good solubility of GNPG3NH2 at pH 14. However, further
studies will be carried out in order to understand this particular
behavior.
the volume distribution peak (Fig. S5, ESIw). Because the DLS
analysis is very sensitive to aggregates, it is able to reveal the
formation of very few aggregates (looking at the intensity
distribution peak), but the amount of aggregates is not
significant enough in order to affect the volume distribution
peak. This same observation has also been reported very
recently by Cho et al. on other dendron-stabilized gold
nanoparticles.52
Second, the stability in the presence of a salt was examined.
Whereas the addition of the salt disturbed the solution
of GNPG3CN, GNPG3NH2 remained very stable up to a
concentration of 1.35 M NaCl (Fig. 7). The plasmon band
remained at 526 nm throughout the salt additions, indicating
no disturbance in the gold nanoparticles’ state (Fig. S6, ESIw).
This high critical coagulation concentration (ccc) is comparable
to some reported pegylated gold nanoparticles,25,72 but without
the need for pegylation.
Third, the stability of the nanoparticles upon drying was
tested. GNPG3CN and GNPG3NH2 were both freeze-dried
and water was added in order to redisperse the nanoparticles.
Again, GNPG3NH2 withstood the drying process and readily
redissolved in water as demonstrated by the very little change
in the UV-visible spectrum (Fig. 8) and in the DLS size (Fig. S7,
ESIw) before and after freeze-drying. The increase in size of
14 nm found for the z-average size of GNPG3NH2 after
lyophilization compares well with the 20 nm increase reported
very recently in the literature for other dendron-stabilized gold
nanoparticles.52 However, GNPG3CN degraded upon lyophi-
lization (no redissolution possible). This large difference in
stability between GNPG3CN and GNPG3NH2 was not
Relative stabilities of GNPG3CN (10) and GNPG3NH2 (11)
The overall stability of the new gold nanoparticle-cored PPI
dendrimers was investigated.
First, the stability in PBS buffer (pH 7.4) was assessed
by addition of the appropriate volume of 10ꢀ PBS to a
solution of 10 or 11 in order to reach a concentration of
1ꢀ PBS. While the solution of GNPG3CN turned blue and
agglomerated (Fig. 6A), probably due to the pH being above
5, the solution of GNPG3NH2 showed great stability and
very little effect on its surface plasmon resonance, as observed
in the UV-visible spectrum in Fig. 6B. The DLS measurements
of GNPG3NH2 in 1ꢀ PBS exhibit some broadening of
the intensity distribution peak but no effect was noticed on
Fig. 7 Stability of GNPG3NH2 toward the presence of a salt: evolution
of the absorbance at the surface plasmon band when increasing NaCl
concentration.
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a JEOL ECX 400 MHz NMR spectrometer. Chemical shifts
are in parts per million (d) using an internal standard to TMS
as the reference, set to 0.00 ppm. ESI mass spectra were
recorded on Bruker Esquire 3000 plus (Billerica, MA). High
resolution mass spectra were recorded on Bruker Daltonics
(Billerica, MA) Apex II Fourier Transform Ion Cyclotron
Resonance equipped with a 12T super conducting magnet with
a micro ESI source (Billerica, MA). Elemental analyses were
performed by Atlantic Microlab, Inc. (Norcross, GA). UV-vis
absorption spectra were recorded on a DU 730 UV/Vis life
science spectrophotometer. Dynamic light scattering (DLS)
data and zeta potentials were obtained by using a Zetasizer
Nano-ZS particle characterization system. Transmission electron
micrographs (TEM) were taken by using a Tecnai T12 electron
microscope equipped with an ATM digital camera. The
pictures acquired were analyzed using SigmaScan Pro software
to obtain the mean size of the gold nanoparticles (150 particles
were measured) and the associated standard deviation. The
Fourier-transform infrared (FTIR) spectra were recorded
using a Paragon 500 FT-IR (Perkin Elmer) instrument
equipped with a ZnSe attenuated total reflectance (ATR)
crystal. A drop of the dissolved gold nanoparticles solution
was cast on the ATR crystal and the FTIR spectra were
recorded after evaporation of the solvent to create a film on
the crystal surface.
Fig. 8 Stability of GNPG3NH2 toward freeze-drying. UV-visible
spectra of GNPG3NH2 before (solid line) and after freeze-drying
and redissolution in water (dashed line).
anticipated, as this goes beyond the difference of solubility. One
possible explanation could be the presence of hydrogen bonds
between primary amines on the same nanoparticle, creating a
more compact layer onto the gold nanoparticles surface.73
Experimental
Materials and methods
All solvents and reagents were purchased from Fisher/Acros
or Sigma Aldrich and used without purification. Anhydrous
solvents were retrieved from the solvent purification system
and stored under a nitrogen atmosphere shortly before use.
Reactions at ‘‘room temperature’’ were conducted at ambient
laboratory temperature (20–26 1C). Distillation of solvents on
a rotary evaporator at 20–50 1C and 25–15 mm Hg is referred
to as ‘‘removal of solvent under reduced pressure.’’ Thin layer
chromatography on silica gel (SiO2) was performed using
Whatman Partisil K5 150 A glass plates (250 mm) without a
preadsorbent zone. Column chromatography was performed
on silica gel (Selecto, 32–63 mesh).
Xanthene-protected mercaptotetraethylene glycol (3). To dry
mercaptotetraethylene glycol 2 (0.209 g, 0.996 mmol) was
added 4% trifluoroacetic acid in anhydrous dichloromethane
(50 mL) and allowed to stir at room temperature for 15 min
under argon. 9-Hydroxyxanthene (0.208 g, 1.05 mmol) was
added to the solution and allowed to stir at room temperature
under argon for 5 h. The solution was concentrated and
the residue was taken up in dichloromethane, washed with
saturated sodium bicarbonate, dried over sodium sulfate,
filtered and concentrated under reduced pressure. The crude
oil was purified via column chromatography (ethyl acetate)
to obtain the product as a yellow oil (0.218 g, 56.1% yield):
Rf = 0.45 (SiO2, Ethyl Acetate); 1H NMR (400 MHz, CDCl3)
d 2.49 (t, 2H), d 3.32 (t, 2H), d 3.44–3.47 (m, 2H), d 3.55–3.71
(m, 11H), d 5.33 (s, 1H), d 7.06–7.12 (m, 4H), d 7.22–7.27
(m, 2H), d 7.44 (dd, 2H); elemental analysis: calcd for
[C21H26O5S, 0.4 H2O]: C, 63.42; H, 6.79; O, 21.72; S, 8.06%.
Found: C, 63.50; H, 6.74; O, 21.71; S, 8.38%.
All glassware used for the gold nanoparticles preparation were
cleaned with freshly prepared aqua regia solution (HCl/HNO3,
3 : 1), then rinsed thoroughly with ultrapure water before use.
The water was distilled and subsequently purified to 18 mO
ultrapure water quality using the Milli-Q academic system. Gold
chloride (HAuCl4ꢁH2O) and trisodium citrate (Na3C6H5O7ꢁ
2H2O), obtained from Electron Microscopy Sciences, were
of analytical grade and were used without further purification.
The centrifugation of the gold nanoparticles was performed
using an Avanti J-E centrifuge (Beckman Coulter) and a JA-20
rotor (Beckman Coulter). The Millex-LCR 0.45 mm syringe
filters were obtained from Millipore. The dialysis membrane
used was a regenerated cellulose dialysis tubing (nominal
MWCO: 12 000–14 000) from Fisher Scientific. The TEM
carbon coated 200 mesh copper grids were purchased from
Ted Pella, Inc.
Xanthene-protected mercaptotetraethylene glycol tosylate (4).
A solution of sodium hydroxide (1.19 g, 29.9 mmol) in
2 mL water was added to a mixture of xanthene-protected
mercaptotetraethylene glycol 3 (3.26 g, 8.34 mmol) in 50 mL
THF. At room temperature, a solution of toluene sulfonyl
chloride (4.84 g, 25.4 mmol) in 15 mL THF was added. After
stirring at room temperature for 24 h, the reaction mixture was
poured in water and extracted with dichloromethane. The
organic layers were combined and dried over sodium sulfate,
filtered and concentrated under reduced pressure. The crude oil
was purified via column chromatography (1: 1 hexane/ethyl
acetate) to obtain the product as a yellow oil (3.99 g, 87.8%
A stock solution of 10ꢀ phosphate-buffered saline (PBS
buffer, pH 7.4) was prepared and 1ꢀ PBS buffer (pH 7.4) was
obtained by dilution with pure water. The pH studies were
carried out using 1 N HCl or 2 N NaOH.
Characterization
1
Proton and carbon nuclear magnetic resonance spectra
(1H NMR and 13C NMR, respectively) were obtained using
yield): Rf = 0.688 (SiO2, 1 : 1 hexane/ethyl acetate); H NMR
(400 MHz, CDCl3) d 2.43 (s, 3H), d 2.47 (t, 2H), d 3.30 (t, 2H), d
c
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3.41–3.44 (m, 2H), d 3.50–3.55 (m, 6H), d 3.64 (t, 2H), d 4.12 (t,
2H), d 5.32 (s, 1H), d 7.06–7.12 (m, 4H), d 7.22–7.26
(m, 2H), d 7.31 (d, 2H), d 7.43 (dd, 2H), d 7.77 (d, 2H);
13C NMR (100 MHz, CDCl3) d 21.74, 28.70, 41.97, 68.75,
69.33, 70.18, 70.56, 70.58, 70.65, 70.81, 116.58, 121.81, 123.59,
128.06, 128.69, 129.72, 129.91, 133.08, 144.88, 152.28; Anal.
calcd for C28H32O7S2: C, 61.74; H, 5.92; O, 20.56; S, 11.77%.
Found: C, 61.67; H, 5.97; O, 20.70; S, 11.77%.
HSTEGG3CN (8). A 20 mL solution of TFA/Et3SiH/CH2Cl2
(2/1/97) was added to 204 mg of dried XanSTEGG3CN
(0.160 mmol) in a 2 neck round bottomed flask under argon.
The reaction mixture was stirred at room temperature for 18 h
and the solvent was evaporated. The residue was washed
with chloroform 3 times, and then dried under high vacuum to
afford light yellow oil (0.170 g, 97%). 1H NMR (D2O):
d (ppm) 7.15–7.13 (d, 2H), 6.88–6.86 (d, 2H), 4.05 (t, 2H),
3.74 (t, 2H), 3.60–3.18 (m, 60H), 2.91 (t, 16H), 2.53 (t, 2H),
2.10 (br s, 12H); ESI [M + H]+ = 1097.7 m/z. HRMS
(ESI) calcd for C58H94N15O4S 1096.73339, found 1096.7560.
IR nmax/cmꢃ1 2248 (nitrile).
Xanthene-protected mercaptotetraethylene glycol bromide (5).
To a stirred solution of xanthene-protected mercaptotetra-
ethylene glycol tosylate 4 (3.99 g, 7.33 mmol) in 75 mL acetone
was added lithium bromide (3.93 g, 45.2 mmol) and was
allowed to stir at reflux for 24 h. The solution was then cooled
to room temperature and concentrated under reduced pressure.
Deionized water was added to the dried compound and was
extracted with dichloromethane. The combined organic
layers were washed with brine, dried over sodium sulfate
and concentrated under reduced pressure. The crude oil was
purified via column chromatography (2 : 1 hexane/ethyl acetate)
to obtain 3.05 g of the pure product as a yellow oil (91.8%):
Rf = 0.589 (SiO2, 2 : 1 hexane/ethyl acetate); 1H NMR
(400 MHz, CDCl3) d 2.49 (t, 2H), d 3.32 (t, 2H), d 3.40–3.47
(m, 4H), d 3.55–3.58 (m, 2H), d 3.59–3.66 (m, 4H), d 3.77
(t, 2H), d 5.33 (s, 1H), d 7.07–7.12 (m, 4H), d 7.23–7.27
(m, 2H), d 7.44 (dd, 2H); 13C NMR (100 MHz, CDCl3)
d 28.71, 30.45, 41.96, 70.21, 70.60–70.71, 71.27, 116.58,
121.82, 123.59, 128.59, 129.73, 152.29; Anal. calcd for
C21H25BrO4S: C, 55.63; H, 5.56; Br, 17.62; O, 14.12; S,
7.07%. Found: C, 55.60; H, 5.53; Br, 17.40; O, 14.36; S, 7.02%.
HSTEGG3NH2 (9). To
a
solution of dendron
7
(XanTEGG3CN) (0.060 g, 0.047 mmol) in 10 mL anhydrous
THF, a borane methyl sulfide complex (0.8 mL, 8.42 mmol)
was injected under argon. The reaction mixture was stirred
at room temperature. The borane methyl sulfide complex
was added again (0.8 mL) after 2 h and after overnight stirring
(0.8 mL). The mixture was allowed to stir for 5 h and methanol
was added slowly until no further bubbling or reaction was
observed. The solution was allowed to stir overnight at room
temperature, after which the solvent was removed under
reduced pressure. Methanol (50 mL) was added to the oily
white residue and the mixture was allowed to stir at reflux
overnight. The solution was then cooled down to room
temperature and concentrated under reduced pressure. The
residue was dissolved in minimum volume of methanol and
precipitated with diethyl ether 3 times to afford colorless oil
1
(0.050 g, 95%). H NMR (D2O): d (ppm) 7.14–7.12 (d, 2H),
6.87–6.85 (d, 2H), 4.06 (br s, 2H), 3.75 (br s, 2H), 3.60–3.4
(m, 10H), 3.22–3.20 (d, 2H), 2.9–2.2 (m, 60H), 1.51 (br s, 28H);
13C NMR (100 MHz, D2O) d 130.02, 115.05, 69.87, 69.71,
69.16, 61.53, 61.29, 60.39, 54.5, 51.20, 51.10, 50.51, 38.66,
XanSTEGG3CN (7). To a solution of dendron 6 (G3CN)
(0.245 g, 0.271 mmol) in 10 mL anhydrous THF, sodium
hydride (60% dispersion in mineral oil) (0.033 g, 0.815 mmol)
was added under argon. After a color change from very pale
yellow to orange (around 3 h of constant stirring), xanthene-
protected mercaptotetraethylene glycol bromide 5 (0.430 g,
0.948 mmol) in 5 mL anhydrous THF was injected to the
deprotonated dendron. The solution turned yellow and was
allowed to stir for 3 days at room temperature, after which
1.5 mL methanol was added. The solvent was removed under
reduced pressure. A small volume of brine was added and the
compound was extracted with chloroform. The combined
organic layers were dried over sodium sulfate, filtered and
concentrated under reduced pressure. The obtained oil was
dissolved in a minimum of chloroform and precipitated by
pouring a large amount of diethyl ether. This reprecipitation
technique was repeated three times in order to remove the
excess of compound 5. After drying under high vacuum, light
yellow oil was afforded (0.345 g, quantitative yield). 1H NMR
(CDCl3): d (ppm) 7.46–7.42 (d, 2H), 7.24–7.22 (d, 2H),
7.11–7.06 (m, 6H), 6.82–6.80 (d, 2H), 5.32 (s, 1H), 4.06–4.04
(t, 2H), 3.8–3.3 (m, 12H), 2.8–2.4 (m, 62H), 1.57 (t, 12H);
13C NMR (100 MHz, CDCl3) d 157.14, 152.21, 132.78, 129.66,
128.76, 128.64, 123.55, 121.81, 118.94, 116.52, 114.60, 70.78,
70.67, 70.50, 70.15, 56.08, 52.13, 51.63, 51.45, 49.59, 41.89,
30.55, 27.88, 24.37, 22.10, 22.00, 14.17. IR
3000–3500w (primary amines).
n
max/cmꢃ1
Citrate gold nanoparticles (citrate GNP)
A solution of gold chloride (HAuCl4ꢁH2O) in pure water
(300 mL, 0.306 mM) was brought to boil. The stirring rate
of the solution was then increased so that a vortex formed. At
that point, trisodium citrate (4.33 mL of 5% citrate solution,
ncit/nAu = 8) was quickly added and the solution was allowed
to stir fast at reflux for 20 min. A series of color changes
occurred after the addition of citrate: the color changed from
very pale yellow (before adding citrate) to colorless (right after
addition), to pink, to progressively more reddish, and finally a
switch to deep ruby red. After 20 min reflux, the solution of
gold nanoparticles was allowed to slowly cool down to room
temperature. UV-vis: lSPR/nm 520. DLS: Z-average size =
19.5 nm; PDI (polydispersity index) = 0.03.
GNPG3CN (10). Before use, a solution of citrate GNP
(25 mL) was centrifuged at 17 400 g for 35 min at 14 1C.
The red pellet was redissolved in pure water and the super-
natant was centrifuged again at 17 400 g for 25 min at 14 1C.
The second pellet was added to the first pellet and the volume
was adjusted to 30 mL with pure water. This solution of citrate
GNP was transferred to an Erlenmeyer and subjected to fast
32.47, 28.77, 25.02, 24.46, 17.24, 16.97. ESI [M + H]+
=
1276.9 m/z. HRMS (ESI) calcd for C71H102N15O5S
1276.79091, found 1276.7967. IR nmax/cmꢃ1 2246 (nitrile).
c
2372 New J. Chem., 2011, 35, 2366–2374
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stirring at room temperature. A solution of HSTEGG3CN 8
(35 mg, 0.032 mmol, 175 equiv./ligand) in 3 mL water was
filtered with a 0.45 mM syringe filter and quickly poured into
the citrate GNP solution under fast stirring. A slight darken-
ing of color occurred after the addition. The mixture was kept
under fast stirring at room temperature for 24 h. The red
solution was centrifuged at 35 000 g for 35 min at 10 1C. The
pellet was redissolved in pure water and dialyzed against pure
water for 24 h (water changed 3 times). The resulting red
solution of GNPG3CN was used as is for characterization.
UV-vis: lSPR/nm 523. DLS: Z-average size = 23.8 nm; PDI
(polydispersity index) = 0.176.
4 I. J. Majoros, C. R. Williams and J. R. Baker, Jr., Curr. Top. Med.
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8 L.-A. Tziveleka, A.-M. G. Psarra, D. Tsiourvas and C. M. Paleos,
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9 G. R. Newkome and C. D. Shreiner, Polymer, 2008, 49, 1–173.
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13 V. Torchilin, Eur. J. Pharm. Biopharm., 2009, 71, 431–444.
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GNPG3NH2 (11). The same overall procedure as for
GNPG3CN 10. 35 mL of citrate GNP was used and a solution
of HSTEGG3NH2 9 (50 mg, 0.044 mmol, 175 equiv./ligand)
in 2 mL water was added. One difference resides in the
pH evolution: after addition of HSTEGG3NH2 9, the pH
increased to slightly basic pH (8–9), so 1N HCl was used to
decrease the pH to around 5, in order to insure optimal
solubility in water. UV-vis: lSPR/nm 525. DLS: Z-average
size = 30.9 nm; PDI (polydispersity index) = 0.144.
19 H.-S. Cho, Z. Dong, G. M. Pauletti, J. Zhang, H. Xu, H. Gu,
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Conclusions
Monodisperse gold nanoparticle-cored PPI dendrimers of
about 20 nm in diameter could be prepared by direct ligand
exchange on citrate GNP. The nitrile terminated dendrimer’s
solubility in water depends on its protonation, and is thus
linked to the pH: GNPG3CN is soluble at pH 4.5 and below,
but flocculates at about pH 5 and above, where its zeta potential
is close to zero. However, the amine terminated dendrimers
remain soluble under basic conditions, up to pH 14. Also,
GNPG3NH2 presents a remarkable stability: it is stable in
1ꢀ PBS buffer, can withstand high salt concentrations, and
can be dried and redissolved without sign of degradation. Such
stability, combined with its ability for lysosomal escape, makes
GNPG3NH2 a candidate of choice as a drug delivery platform.
Finally, the primary amine termini of PPI dendron 9 provide
great convenience for direct derivatization with moieties such as
drugs or targeting entities, for the formation of multifunctional
drug delivery systems with high payloads.
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Acknowledgements
The authors are grateful for the financial support from the
AACR/PanCAN (Career Development Award, grant no.
08-20-25-DANI) and from the Department of Defense (Prostate
Cancer Research Program, CDRMP, grant no. PC081299).
They also would like to acknowledge Josh Wilhide for the
acquisition of the HRMS spectra.
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