Affinity purification of multifunctional polymer nanoparticles

Article abstract of DOI:10.1021/ja1058982

We report that multifunctional polymer nanoparticles approximately the size of a large protein can be 'purified', on the basis of peptide affinity just as antibodies, using an affinity chromatography strategy. The selection process takes advantage of the thermoresponsiveness of the nanoparticles allowing 'catch and release' of the target peptide by adjusting the temperature. Purified particles show much stronger affinity (K_dapp ≈ nM) and a narrower affinity distribution than the average of particles before purification (K_dapp > μM) at room temperature but can release the peptide just by changing the temperature. We anticipate this affinity selection will be general and become an integral step for the preparation of "plastic antibodies" with near-homogeneous and tailored affinity for target biomacromolecules.

Full text of DOI:10.1021/ja1058982

Published on Web 09/09/2010
Affinity Purification of Multifunctional Polymer Nanoparticles
,†
†
‡
†
§
Yu Hoshino,* Walter W. Haberaecker III, Takashi Kodama, Zhiyang Zeng, Yoshio Okahata, and
,
†
Kenneth J. Shea*
Department of Chemistry, UniVersity of California, IrVine, California 92697, Department of Mechanical
Engineering, Stanford UniVersity, Stanford, California 94305, and Department of Biomolecular Engineering,
Tokyo Institute of Technology, Yokohama 226-8501, Japan
Received July 4, 2010; E-mail: yhoshino@chem-eng.kyushu-u.ac.jp; kjshea@uci.edu
Abstract: We report that multifunctional polymer nanoparticles
approximately the size of a large protein can be “purified”, on
the basis of peptide affinity just as antibodies, using an affinity
chromatography strategy. The selection process takes advantage
of the thermoresponsiveness of the nanoparticles allowing “catch
and release” of the target peptide by adjusting the temperature.
Purified particles show much stronger affinity (Kdapp ≈ nM) and a
narrower affinity distribution than the average of particles before
purification (Kdapp > µM) at room temperature but can release
the peptide just by changing the temperature. We anticipate this
affinity selection will be general and become an integral step for
the preparation of “plastic antibodies” with near-homogeneous
and tailored affinity for target biomacromolecules.
Figure 1. (a) Amino acid sequence of melittin. Hydrophobic and positive
charged residues are printed in green and red respectively. (b) The chemical
structures of functional monomers used for NP synthesis. (c) Solution phase
AFM images of NPs. A height profile of cross section (light blue line) is
shown in insert.
(
TBAm) and negatively charged acrylic acid (AAc) functional
monomers (Figure 1b). We have reported that NPs with this
composition interacts with melittin (Kdapp ) 46 µM) via both
electrostatic and hydrophobic interactions in PBS (35 mM phosphate
5
General procedures for the creation of synthetic materials with
biomacromolecular recognition sites are of significant interest as a route
buffer/0.15 M NaCl, pH 7.3). However, the melittin affinity of
the NPs exhibits a significant medium effect; there is only a modest
affinity for melittin in low salt containing media such as water.
The medium effect may be attributed to hydrophobic interactions
between melittin and the NPs that are enhanced by the salting out
1-8
to stable, robust, and mass-produced substitutes for antibodies.
Ideally, recognition of complex biological targets, including
proteins, peptides, and carbohydrates, requires multiple functional
groups that contact target molecules by a combination of electro-
static, hydrogen-bonding, van der Waals, and/or hydrophobic
interactions. It has been shown that copolymerization of optimized
combinations and ratios of functional monomers creates synthetic
1
2
effect of sodium phosphate and chloride ion.
NPs were synthesized by a free radical copolymerization. To
quantify the concentration of the NPs by fluorescence and to monitor
the hydration of polymer chains by the solvatochromic shift of λem,
N-[2-[[[5-(dimethylamino)-l-naphthalenyl]sulfonyl]-amino]ethyl]-
2-methyl-2-propenamide was synthesized and incorporated (1 mol
1
-5
polymer materials with molecular recognition sites.
However,
in contrast to antibodies whose exact sequence can be determined
and cloned, polymerized materials result in heterogeneous structures
1
3
%) into the NPs (Supporting Information). The concentrations
of total monomer, cross-linker, and surfactant (sodium dodecyl
sulfate) were optimized to adjust the size of the NPs to ap-
1
,3,7
with a distribution of recognition sites.
This is an intrinsic
property of polymers synthesized under kinetic control, in contrast
to the synthetic small molecular hosts prepared by multistep
5
,8,14
proximately 30 nm.
%) and carboxylic acid (2.3 mol %) group in the MFNPs was
Incorporation of the tert-butyl (41 mol
9
10
reactions or by self-assembly under equilibrating conditions. Here
we demonstrate a general procedure to purify synthetic polymer
nanoparticles (NPs) with high-affinity binding sites for a target
biomacromolecule from a random pool of multifunctional copoly-
mer nanoparticles (MFNPs). These nanoparticles are approximately
the size of a large protein and are “purified” on the basis of peptide
affinity as in the case of antibodies, using an affinity chromatog-
raphy strategy.
1
13
13
quantified by H NMR and inverse-gated C NMR utilizing C-
enriched AAc (Supporting Information). NP size was determined
by DLS and AFM (Figure 1c).
The protocol for affinity sorting is shown in Figure 2a. To sort
MFNPs on the basis of melittin affinity, we immobilized melittin
on avidin agarose beads thru an avidin-biotin interaction (Sup-
porting Information). NPs are first incubated with melittin-agarose
beads for 2 h at 25 °C. Here, 100 mM phosphate buffer was used
as the NP adsorption media since interactions between melittin and
NPs in water were too weak to isolate sufficient amounts of NPs
for study (Supporting Figure 1). The supernatant was then separated
from the beads, and the concentration of NPs remaining in the
supernatant was quantified by fluorescence. When NPs (12 µg
The concept of affinity purification of NPs was demonstrated
with melittin, a 26 amino acid peptide (Figure 1a), as the target
molecule. Melittin has six positive charges of which four are
localized in a hydrophilic 6 amino acid sequence on the C-terminus.
The remaining 20 amino acids have a high proportion of apolar
1
1
residues.
-1
For the MFNPs, we chose cross-linked N-isopropylacrylamide
NPs (∼30 nm) incorporating hydrophobic N-tert-butylacrylamide
mL ) were incubated with different volumes of melittin-im-
mobilized beads, the amount of NPs that were captured by the beads
depended on the volume of melittin beads when the volume of beads
†
University of California.
-1
‡
was less than ∼20 µL mL . However, when more than ∼20 µL
Stanford University.
§
-1
Tokyo Institute of Technology.
mL of beads were incubated, no further capture of NPs was
1
3648 9 J. AM. CHEM. SOC. 2010, 132, 13648–13650
10.1021/ja1058982  2010 American Chemical Society

C O M M U N I C A T I O N S
eluted from agarose beads that were not functionalized with melittin
(control beads). A QCM experiment revealed that the NPs that were
released by this cold elution were found to strongly interact with
melittin at 25 °C in pure water (Kdapp ) 0.66-2.3 nM, Figure 2d
insert). This affinity is comparable to a typical antibody-antigen
interaction. In addition, the affinity switching was reversible through
5
cycles of cooling (1 °C) and warming (25 °C). Cold elution was
observed from melittin-immobilized beads for the first few elution
cycles (12 h/cycle). The amount of NPs released after the third
elution cycle however was dramatically lower, and no NPs were
detected after the sixth elution cycle (Figure 2d). This establishes
that the affinity for melittin could be dramatically lowered by
cooling to 1 °C resulting in their release from the agarose beads.
The yield of NPs in the cold elution step is several percent, but
importantly they showed a significantly stronger affinity for melittin
than the NPs before affinity sorting. (Note: the interaction measured
by QCM in water was not observed before sorting (Figure 2d
insert).) To compare the affinity distribution of NPs in solution
before sorting and those released by cold elution, each batch of
NPs (240 ng mL- ) was incubated with melittin immobilized
beads in water at 25 °C overnight. Although only a portion of
the NPs were captured from the NPs before sorting, all of the
NPs in the cold elution were recaptured by melittin-immobilized
beads (Supporting Figure 3). This result indicates that the affinity
distribution of the NPs released by cold elution is narrower than
the NPs before sorting.
Figure 2. (a) Protocol for affinity sorting. Randomly copolymerized NPs
are incubated with melittin immobilized agarose beads in 100 mM phosphate
buffer (pH 7.4) at 25 °C. Ratio of NPs remaining in solution after incubation
with different volumes of melittin immobilized beads is plotted in (b).
Interaction (QCM) between melittin and NPs before incubation (yellow)
and remaining after incubation (gray) is shown in (b) insert. NPs on the
beads were first washed with water at 25 °C and then eluted with cold
water (1 °C). Each cycle (12 h incubation) was repeated until no further
elution of NPs was observed. Fluorescent intensity of NPs in each fraction
from washing (red) and cold elution (blue) cycles is plotted in (c) and (d)
respectively. Interaction between melittin and wash fraction in 100 mM
phosphate buffer (red circles) and in water (red triangles) at 25 °C is shown
in (c) insert. Interaction between melittin and NPs before incubation (yellow)
and cold elution (blue) in water at 25 °C is shown in (d) insert.
1
From these results, we conclude that synthetic polymer nano-
particles with a high affinity for the peptide melittin can be
“purified” from a random pool of multifunctional copolymer
nanoparticles by an affinity chromatography strategy. Each fraction
isolated during the affinity sorting process shows a different affinity
for the target peptide. The “selected” NPs have a much stronger
and narrower affinity distribution than the materials before
purification. We anticipate this affinity purification is applicable
for most nanosize materials for molecular recognition including
observed; approximately 40% of the NPs remained in the super-
natant (Figure 2b). The interaction between melittin and NPs before
incubation or the unbound fraction remaining in the supernatant
after incubation was analyzed by 27-MHz QCM in 100 mM
5
,8,15
phosphate buffer.
The QCM results showed that MFNPs in
the unbound fraction did not interact with melittin, although the
MFNPs before incubation showed a modest interaction with melittin
under the same conditions (Figure 2b insert). This indicates the
as-synthesized MFNPs are comprised of a heterogeneous mixture
of particles, of which some have melittin affinity under the binding
conditions (100 mM phosphate) and others do not.
8
17
molecularly imprinted NPs and functionalized inorganic NPs
and will become an integral step for the preparation of “plastic
antibodies” with a near-homogeneous and tailor-made affinity
for target molecules.
Melittin immobilized beads that were incubated with NPs (120
-1
µL mL ) were washed extensively (12 h/cycle) with pure water
at 25 °C. During the first few cycles, quantities of NPs were washed
from the beads. However, the concentration of NPs diminished with
every washing cycle and no NPs were detected after the eighth
cycle. QCM revealed that the NPs from this wash fraction rebound
with melittin in 100 mM phosphate buffer but little interaction was
observed in water (Figure 2c insert), indicating NPs that recognize
melittin in 100 mM phosphate buffer but not in water were removed
from the beads in this washing step (Figure 2c).
Acknowledgment. We thank Dr. T. Ozeki at Initium, Inc. for
the QCM measurement, D. Gatanaga and Dr. S. Takahashi for the
fluorescent measurements, and financial support from the National
Institutes of Health (GM080506).
Supporting Information Available: Experimental procedures and
supporting data. This material is available free of charge via the Internet
at http://pubs.acs.org.
In the general protocol for affinity purification of proteins,
isolated molecules on the solid affinity support can be released by
selective elution induced by either pH and/or salt gradients or by
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