Isolation and characterization of dendrimers with precise numbers of functional groups

Article abstract of DOI:10.1002/chem.201001175

(Figure Presented) Precision dendrimer: Nine different dendrimer components with precise numbers of a functional ligand were isolated leading to an order of magnitude improvement in the purity of each component.

Full text of DOI:10.1002/chem.201001175

COMMUNICATION
DOI: 10.1002/chem.201001175
Isolation and Characterization of Dendrimers with Precise Numbers of
Functional Groups
[
b, c]
[a]
[c]
Douglas G. Mullen,
Emilee L. Borgmeier, Ankur M. Desai,
[a] [c]
[
a]
[c]
Mallory A. van Dongen, Mark Barash, Xue-min Cheng, James R. Baker, Jr., and
[
a, b, c]
Mark M. Banaszak Holl*
Substantial attention has been devoted to nanoparticles
conjugated with functional ligands. Application of these ma-
terials has included building blocks for nano-scale struc-
Significant progress has been made towards precisely con-
trolled materials using gold nanoparticles. A number of
strategies exist in the literature to synthesize and/or isolate
milligram quantities of gold nanoparticles with a single func-
[1]
[2]
tures, materials for sensing and detection, platforms for
[3]
[4]
[6]
targeted delivery, imaging and diagnostic systems, and
tional group and up to 95% purity has been achieved.
[5]
probes of biological structure. One of the challenging fea-
tures of these systems is the heterogeneous distribution of li-
gands per particle. For the vast majority of systems reported,
these distributions are not characterized nor are they incor-
porated in design parameters. The implications of this heter-
ogeneity are two-fold: First, mixtures containing many
ligand/particle ratios make studies that investigate composi-
tion–activity relationships complex; second, production of
materials with a consistent distribution of ligand/particle
ratios is problematic because of inconsistencies in the nano-
particle preparations and reaction kinetics. New methods
that exhibit precise control over the number of ligands per
particle have the potential for dramatically improved func-
tional efficacy, batch reproducibility, and an enhanced abili-
ty to probe the relationship between activity and the
number of ligands conjugated to a particle.
Gold nanoparticles with 0–5 conjugated ligands have also
been isolated in sub milligram quantities using either gel
[7]
electrophoresis or ion-exchange chromatography.
This level of control has not been attained for other nano-
particle-based systems. In fact, with few exceptions, the ana-
lytical techniques commonly used to characterize these sys-
tems (NMR, HPLC, GPC, MALDI-TOF, UV/Vis) have
been unable to identify the distribution of nanoparticle-
ligand components.
Recently, we have developed the ability to resolve the dis-
tribution of components using HPLC for poly(amidoamine)
(PAMAM) dendrimer samples conjugated with 3-(4-(prop-
[8]
2-ynyloxy)phenyl)propanoic acid (alkyne ligand). The re-
sults of these studies revealed that dendrimer–ligand distri-
butions are heterogeneous (a sample with a ligand mean of
5.7 was composed of 18 dendrimer–ligand components), are
poorly represented by the arithmetic mean, and are sensitive
to pre-existing distributions of conjugation sites on the
parent dendrimer.
[
a] E. L. Borgmeier, M. A. van Dongen, M. Barash,
Prof. M. M. Banaszak Holl
We have now employed semi-preparative HPLC to suc-
cessfully isolate nine different dendrimer components with
precise numbers of ligands. Generation 5 (G5) PAMAM
dendrimer was conjugated with (3-(4-(2-azidoethoxy)phe-
nyl)propanoic acid) (azide ligand) to produce dendrimer
with a mean of 4.3 ligands (Scheme 1). Dendrimer samples
with 0, 1, 2, 3, 4, 5, 6, 7 and 8 azide ligands were isolated
from the dendrimer conjugate 3 and characterized by
H NMR and analytical HPLC. Levels of purity for these
samples were found to be greater than 80%.
Isolation of practical quantities was achieved by carrying
out the HPLC process for 12 runs. Figure 1a shows the
semi-preparative HPLC traces along with grey lines demark-
ing the fractions that were collected every 4 s. A peak-fitting
Department of Chemistry, University of Michigan
9
30 N. University, Ann Arbor, MI 48109 (USA)
Fax : (+1)734-763-2283
E-mail: mbanasza@umich.edu
[
b] D. G. Mullen, Prof. M. M. Banaszak Holl
Macromolecular Science and Engineering, University of Michigan
9
30 N. University, Ann Arbor, MI 48109 (USA)
[
c] D. G. Mullen, A. M. Desai, Prof. X.-m. Cheng, Prof. J. R. Baker, Jr.,
Prof. M. M. Banaszak Holl
Michigan Nanotechnology
Institute for Medicine and Biological Sciences
University of Michigan, 9220 MSRB III
1
1
150 West Medical Center Dr.
Ann Arbor, MI 48109 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201001175.
Chem. Eur. J. 2010, 16, 10675 – 10678
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10675

sample traces before purification. A normalized trace of 3 is
included for reference. The peak area for each of the sam-
ples directly relates to the amount of material that was iso-
lated because samples were characterized at the isolated
concentration. Following purification, the samples were
characterized again by analytical HPLC (Figure 2b) and
1
H NMR spectroscopy.
Three main features stand out in Figure 2. First, each iso-
lated component has the same retention time as its original
position in the distribution. Second, smaller peaks can be
seen adjacent to the major peak in each sample. These
smaller peaks have retention times consistent with other
dendrimer–ligand components. The purity levels of the iso-
lated samples (Table 1) were quantified by peak fitting (Fig-
ure 2c). Finally, no differences were observed in the HPLC
traces before and after purification indicating that the sam-
ples did not degrade during the purification process.
Scheme 1. a) Alkyne ligand, NHS, EDC, acetonitrile, water, RT, 12 h,
9% yield; b) acetic anhydride, triethylamine, MeOH, RT, 12 h, 61%
yield. Detailed experimental methods can be found in the Supporting In-
formation.
7
analysis determined the retention time of each component
(
Figure 1b), thereby identifying the fractions in Figure 1a to
NMR is the second technique used to characterize the iso-
lated samples. Figure 3 shows the H spectrum for the
sample with one ligand. Two different methods were used to
calculate the ligand/dendrimer ratio for each sample
(Table 1).
The first method has two assumptions: 1) All dendrimer
end groups are either acetyl groups or ligands. 2) The mean
number of end groups per dendrimer after HPLC isolation
is 112. Method 1 uses the integrals for the aromatic ligand
protons (aa’ and bb’), normalized by the number of these
protons per ligand (4), divided by the numerator plus the in-
tegral for the methyl protons at 1.9 ppm normalized by the
number of protons per acetyl group. The product (the ratio
of ligands to the total number of end groups per dendrimer)
is multiplied by 112 to yield the number of ligands per den-
drimer.
1
combine for dendrimer samples with 0–8 ligands. These frac-
tions are highlighted in Figure 1a by solid colored bars. The
mass isolated by this process is listed in Table 1.
Analytical HPLC was used to characterize the samples
both before and after purification. Figure 2a displays the
Overall, there is good agreement between the ratios de-
termined by HPLC and by NMR method 1. For samples 5–
8
, the difference between these values is 3% or less. Sam-
ples with 1–4 ligands, however, have differences between 13
and 26%. These larger differences suggest that the assump-
tions in Method 1 may not be appropriate. In addition to 3
being composed of a distribution of dendrimer/ligand ratios,
the dendrimer alone is a polymer with different backbone
structures. Considering that a small section of each compo-
nent peak was isolated from the distribution in Figure 1a,
the isolated dendrimer particles in each sample may not
have a mean of 112 end groups.
Using a different set of assumptions, a second method was
used to calculate the NMR ratios. This method uses the pro-
tons from the interior of the dendrimer as a reference. The
reference integral was determined using 4 which was also
1
made from 1. In the H NMR spectrum for this material, the
integral of the methyl protons c was normalized to 336. This
provided the number of interior protons, f, g, and e per den-
drimer. These protons were then used as an internal reference
to quantify the ligand/dendrimer ratio in the isolated samples.
Method 2 assumes that all amine groups in 4 were acety-
lated and that the number of interior protons is not sensitive
to the isolation process. The ligand/dendrimer ratios, calcu-
Figure 1. Isolation of dendrimer–ligand components by semi-preparative
HPLC. a) Semi-preparative HPLC traces for the 12 identical runs. The
1
20 fractions starting at 20 min are shown in grey. The selected fractions
for each of the different dendrimer–ligand components (0–8) are high-
lighted in solid colored lines. b) Peak fitting analysis of the trace for Run
6
. The HPLC data is presented in red circles and the multiple copies of
the fitting peak are shown in green. The summation of these fitting peaks
6
is shown in blue. The residual values in panel b are multiplied by 10 .
10676
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 10675 – 10678

Dendrimers
COMMUNICATION
Table 1. Characterization of isolated dendrimer with precise numbers of ligands.
Nominal no. of
ligands per den-
drimer
Mass
recovered
[mg]
HPLC Analysis
NMR Analysis
Method 1
No. ligands per
dendrimer
Purity
[%]
Increase in
purity
Method 2
[
a]
[b]
No. ligands per
dendrimer
Difference
[%]
No. ligands per
dendrimer
Difference
[%]
[
c]
[c]
0
1
2
3
4
5
6
7
8
10.4
10.1
8.5
9.5
8.7
5.4
6.7
1.5
1.8
0.0
1.0
1.9
2.7
3.8
4.8
5.8
6.7
7.7
100
97
93
88
86
84
84
82
79
14ꢁ
6ꢁ
6ꢁ
7ꢁ
9ꢁ
10ꢁ
12ꢁ
14ꢁ
16ꢁ
0
0
0
0
1.2
2.4
3.4
4.3
4.9
5.7
6.8
7.8
20
26
26
13
2
2
3
1
1.1
2.2
3.2
3.9
4.5
5.2
5.8
6.9
10
14
17
3
7
10
13
10
[
a] Defined by the number of ligands per dendrimer. [b] Fold increase in purity is relative to the amount of the component in the distribution (dendrimer
3
). [c] Difference is calculated by the NMR ratio minus the HPLC ratio, divided by the HPLC ratio.
Figure 2. Analytical HPLC analysis for the isolated dendrimer–ligand components. a) Baseline-corrected traces for dendrimer–ligand components with
–8 ligands run immediately after the isolation process. The area of each peak is directly proportional to the amount of isolated material. The HPLC
0
trace (normalized) for the dendrimer distribution with a mean of 4.3 ligands is also included. b) Traces for the isolated dendrimer–ligand components
after purification (each trace baseline corrected and normalized). Blue lines show relationship between the isolated component and the material with the
distribution of components. In addition to the major peak in each component, small amount of other components have been detected. c) The peak fitting
method was used to quantify the purity of each isolated component. Fitting peaks are shown in green, with the summation of these peaks in blue. The
6
HPLC data is shown in red circles. The residual values in panel c are multiplied by 10 .
Chem. Eur. J. 2010, 16, 10675 – 10678
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10677

M. M. Banaszak Holl et al.
gands. Peak fitting analysis on analytical HPLC traces deter-
mined the sample purity to be 80% or higher. The number
1
of ligands per dendrimer, quantified by H NMR spectrosco-
py, were in good agreement with the number quantified by
HPLC and peak fitting. Significantly, for dendrimer com-
pounds with 0–4 ligands, over 8 mg of material was pro-
duced in this study. This approach shows great promise to
overcome batch reproducibility challenges in dendrimer-
based systems.
Acknowledgements
This work was supported in part with Federal funds from the National
Cancer Institute, National Institutes of Health, under Award 1 R01
CA119409 and Department of Defense DARPA award W911NF-07-1-
0
437.
Keywords: dendrimers · drug delivery · ligand distribution ·
nanoparticles · nanotechnology
1
Figure 3. H NMR characterization. a) Spectrum for the isolated dendri-
mer with 1 ligand. b) Chemical structure and proton labels for the azide
ligand and acetyl-terminated dendrimer arms.
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4
2–47.
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In conclusion, we have successfully employed a semi-
preparative HPLC to isolate dendrimer with 0–8 azide li-
Received: May 3, 2010
Published online: August 3, 2010
10678
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 10675 – 10678