Synthetic Polymer Nanoparticles: Abiotic
Receptors for Peptides, Proteins and
Carbohydrates
45th Annual Oak Ridge Conference
April 18-19, 2013
Baltimore, Md.
Kenneth J. Shea
Department of Chemistry &CEMS
University of California, Irvine
Irvine, California
Synthetic Polymer Nanoparticles. Abiotic Receptors
for Peptides, Proteins and Carbohydrates.
Staphylococcal
enterotoxin B
Conserved
regon
GFP
Human IgG1
hydrophilic residues
hydrophobic residues
gp120
Melittin
glycoprotein
Outline
•Introduction to research goals
•Lessons from biology
•Research strategy
•“Plastic” antibodies
•ELISA mimic-expansion of monomer pool
•Catch and release of proteins
•Protein A mimic
1
Goal: Develop a general strategy for preparing
synthetic polymers with antibody-like affinity
and selectivity for peptides, proteins and
carbohydrates.
Polymer-Ligand
Conjugate
Polymer-Antibody
Conjugate
Target Protein
or Peptide
Lessons to Learn from Protein-Protein Interactions
SARS Protein Attaching to a Host Cell Receptor
Recognition results from the cumulative effect of many individually
weak interactions over a surface area of 300 - >1000 A2
Stephen C. Harrison, HHMI, http://www.childrenshospital.org/cfapps/research/data_admin/Site142/mainpageS142P4.html
Protein-Polymer
Interactions?
HO
-O
2C
CH3
HO
CH3
CH3
Protein-Protein
Interactions
CH3
Polymer
Nanoparticle
2
Target Peptide = Imprinting Peptide
• Melittin; cytotoxic 26 amino
acid peptide, from honey bee
venom
•
Amphiphilic
•
Pore forming toxin, creates unregulated
Video removed
pores in the membrane of targeted cells
Nanoparticle Library
Precipitation Polymerization
nanoparticle library
composition
Color coded distribution of
monomers used for polymer synthesis
Interaction Between Co-polymers and Target Peptide
Melittin”
Hemolysis Neutralization Assay
Both charged (-) and hydrophobic
groups are necessary
3
Nanoparticle Functional Monomer Composition
vs RBC Neutralization Constant
Final Step-Melittin Imprinting
Inhibition of Hemolytic Activity by MIP NPs
Capacity of Melittin Neutralization
Video removed.
Melittin
 Hemolysin
Binding capacity of
imprinted nanoparticle
11  mol / g
Binding capacity of IgG
13.4  mol / g
Small., 5, 1562-1568 (2009).
Are Melittin Specific Imprinted NPs
Effective in a Biological Milieu?
In Vitro Neutralization of Melittin Activity
(Inhibition of melittin induced cytotoxicity in cultured
cells by MIP/NIP)
In Vivo Studies
(Neutralization of Melittin Toxicity in Mice)
12
4
In Vitro Studies
Inhibition of Cytotoxicity
Inhibition of cytotoxicity of cultured HT-1080 human fibrosarcoma
cells by NPs is dose dependent.
13
Therapeutic Polymer Nanoparticles?
Can We Use Polymer NPs as a Toxin Antidote?
Determination of survival rate of mice after intravenous
injection of melittin w/o polymer NPs
J. Am. Chem. Soc. 2010, 132, 6644-6645
14
Survival of Melittin-Challenged Mice Followed by
Injection of MIP NPs.
Survival curves of mice over 24 hrs after
injection (IV) of 5.0 mg kg-1 melittin with
9.4 mg kg-1 (blue), 30 mg kg-1 (red), and
without MIPNPs (green).
Surviving rate of mice 24 h after melittin
injection with/without (blue triangles/green
circles) 9.4 mg kg-1 MIPNPs
This shift is close to what was expected by
the neutralization capacity experiment of
hemolytic activity.
15
5
Biodistribution of NPs
Fluorescein Labeled PNPs
0.5 % Fluorescein Acrylate
Cy5 labeled Melittin
(via amine on N-term Lys)
Excitation; 467 or 482 nm
Emission; 515 nm
Excitation; 649 nm
Emission; 666 nm
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Biodistribution of NPs
In vivo imaging with fluorescent labeled melittin and molecularly imprinted nanoparticles
(MIPNPs) establishes the MIPNPs accelerate clearance of the toxic peptide (melittin)
from blood. The MIPNP peptide complex accumulates in the liver.
Biodistribution of NPs
NPs and melittin were labeled with different
dyes to monitor localization in liver.
Fluorescence microscopy of liver tissue
Cy5-Melittin
Fluorescein-MIPNPs
Cy-5 dye
labeled melittin
(via epsilon
amine on
additional Lys
on the N-term)
Fluorescein Acrylate
(0.5%) was incorporated
in MIPNPs
Excitation; 467& 482 nm
Emission; 515 nm
Excitation; 649 nm
Emission; 666 nm
PNAS, 2012, 109, 33-38
6
Affinity Sorting of Plastic
Antibodies
Synthetic polymer NPs are not homogeneous materials.
They are crude polyclonals with a distribution of sizes and
affinities.
However, since they are nano size, they can be
separated on the basis of affinity just as antibodies and
other large proteins
Temperature Effect on Nanoparticle-Melittin
Binding (LCST)
25°C
10°C
Temperature Effect on Nanoparticle-Melittin
Binding
12°C
25°C
LCST ~15°C
NIP
MIP
Melittin release at low temperature.
Affinity Selection of NPs
Thermal Catch and Release
Random
Copolymer
Nanoparticles
(NPs)
Peptide Immobilized
Agarose Beads
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Affinity Selection of NPs
Thermal Catch and Release
Random
Copolymer
Nanoparticles
(NPs)
Peptide
PeptideImmobilized
Immobilized
Agarose Beads
Agarose
Beads
J. Am. Chem. Soc. 2010, 132, 13648-13650
Polymer-NP Binding-What’s Important?
Hydrogen bonding
Electrostatic interactions
Hydrophobic interactions
• Synthetic Polymer NP-Polysaccharide Interactions:
A Systematic Study by ITC
JACS, 2012, 134, 2681 - 2690.
A Systematic Study of Polymer NP-Polysaccharide
Interactions
• Binding Forces involved in the NP‐Polysaccharide Interactions
• Factors (pH, ionic strength, temp) that affect the NP‐
polysaccharide interactions
• Potential roles of NPs in inhibition of polysaccharide‐
protein interactions
8
Nanoparticle Synthesis
Heparin
Text
a polyanionic carbohydrate
JACS, 2012, 134, 2681 - 2690.
25
Thermodynamic Study: Heparin-NPs
Syringe: Heparin
Reference Cell
Sample
Cell: NPs solution
Neutral polyAAm
5% AAC
5% ATC
Entry
N
Ka (106M‐1)
Δ H
(kcal/mol)
ΔS (cal/mol/deg)
#1(90%AAm)
0.40 ± 0.14
0.8±0.3
‐51.1±19.8
‐144
#2(5%AAc)
‐
‐
‐
‐
#4(5%ATC)
8.63 ± 0.12
2.10± 0.25
‐52.7 ±1.0
‐148
Thermodynamic Study:
Heparin-Positive Charged NPs
2% ATC
2% APM
Entry
#3 (2% ATC)
N
3.86 ± 0.05
5% ATC
5% IM
Ka (106M‐1)
1.48±0.08
Δ H
(kcal/mol)
‐56.2±1.0 ΔS (cal/mol/deg)
‐161
‐148
#4(5%ATC)
8.63 ± 0.12
2.10± 0.25
‐52.7 ±1.0
#5(10%ATC)
16.2±0.28
21.2±6.77
‐37.0±1.1 ‐91
#6(2%APM)
2.35 ±0.03
1.52±0.05
‐75.4±1.1
‐225
#8(5% IM)
1.99± 0.05
2.25± 0.20
‐154.0± 5.1
‐489
#9(5%AEM)
3.18± 0.03
1.64± 0.06
‐97.4± 1.2
‐298
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Flexibility of Polymer Chain: Crosslinking Degree of NPs (2%ATC)
VS
Polymer Main Component: AAm vs. NIPAm
Enthalpy Driven
Ionic + H-bonding
5% ATC (AAm)
Entropy Driven
Ionic interactions
little H-bonding
5% ATC(NIPAM)
Heat capacity change
Inhibition of Heparin-Protein Interactions by NPs
Anticoagulant Assay
10%ATC
5%ATC
2%ATC
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Conclusions
• Hydrogen bonding, electrostatic interactions and dehydration from polar
patches are involved in the NP-polysaccharide interactions. Hydrophobic
interactions are less important.
• Charge density, nature of charged groups, cross-linking and the capacity
for hydrogen bonding are critical for high NP affinity. Select NPs can
inhibit the heparin-protein interaction.
ELISA-Mimic Screen for Synthetic Polymer
Nanoparticles with High Affinity toTarget Proteins
Biomacromolecules, 2012, 13, ASAP
Functional Monomers for NP Synthesis
Which functional groups are best
for a specific protein?
11
ELISA-Mimic Screen for Synthetic Polymer NPs
hydrophobic, pI =11
hydrophobic, pI = 5.5
Solution Phase Separation Efficiency
Temperature Effect on Nanoparticle Structure
(LCST)
25°C
10°C
36
12
Engineered Protein Catch and Release
Depletion of Lysozyme from Solutions at RT
Binding selectivity of NP2 at room temp
Recycling - Protein Retains Activity After Capture and
Release
Temperature-responsive “catch-and-release” of lysozyme (5 mgmL1) by NP2
(2.0 mg). Experiments were carried out in PBS (sodium phosphate buffer (35
mm) containing NaCl (150 mm), pH 7.3).
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Protein Composition of Egg White
Egg White contains approximately 40 different proteins
54% Ovalbumin - Nourishment; blocks
digestive enzyme
12% Ovotransferrin - Binds iron
11% Ovomucoid - Blocks digestive enzymes
4% Ovoglobulin G2
4% Ovoglobulin G3
3.5% Ovomucin
3.4% Lysozyme
1.5% Ovoinhibitor
1% Ovoglycoprotein
0.8% Flavoprotein
0.5% Ovomacroglobulin
0.05% Avidin
0.05% Cystatin
(Total listed 95.8%)
Selective Protein Catch and Release
Lysozyme Capture from Egg White.
Angewandte Chemie, 2012, 51, 2405–2408
lysozyme
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Can a synthetic polymer NP be engineered to have
an intrinsic high affinity to a specific domain of a
large biomacromolecule?
JACS, 2012, 134, ASAP
Development of a Plastic Protein A
Protein A
Target Protein IgG
Fab
Fragment
Fc
Fragment
Proteins A and G are
used to purify IgG in
on a commercial scale
150 kDa
More than 10,000 kg of IgG
are purified a year.
Library of Multifunctional Polymer NPs
AAc
Free Radical
Can we prepare synthetic NPs to
capture the Fc fragment of IgG?
TBAm
Surfactant
Plastic Protein A ?
NIPAm
APM
~50 nM
Nanoparticles
The interactions between NP7 and IgG, BSA, TNFα and hemoglobin in each of
which were immobilized on a QCM surface in PBS (35 mM, pH 7.3 with 150 mM
NaCl)
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Reproducibility ?
The interaction between NP7 and the Fc fragment in water. Red and Black are NP7 from different
batches. The squares plot interactions between NP7 and Fc fragment and the circles plot the
interaction between NP7 and the QCM gold surface under the same conditions except the Fc fragment
was omitted. It demonstrated that implied that the interaction is not due to batch specific
pH Dependence of NP-IgG Affinity
Compitition Studies with Protein A
Protein A binding site shown on IgG1–Fc.
Binding site atoms are colored cyan.
Computationally selected NP binding sites at (a) neutral and (b) low pH. Computational results
indicate NP7 is more likely to bind the protein A binding site at lower pH in agreement with
experimental observations. Residue side-chains are colored green if the combined score of its surface
region is greater than 0.0, and brighter shades of green indicate higher scores
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Binding isotherm of Fc domain to NP7 immobilized
on QCM surface
Water (pH 5.5) Standard deviations were calculated from three independent measurements.
Conclusions
NPs that bind to Fc fragments of IgG were identified from a library of
multifunctional polymer NPs.
NP binding domain(s) on the Fc fragment were identified from the Fc-protein A
crystal structure and competition binding experiments.
Affinity could be controlled by adjusting pH of the solution.
NP binding constant (at pH 5.5) was comparable to protein A (at pH 7.2), but
the binding capacity was 5 times smaller than protein A.
These results suggest that synthetic polymer NPs can be
engineered to have an intrinsic affinity to a specific domain of
a large biomacromolecule.
Acknowledgements
UCI
Dr. Hidekazu Nishino
Dr. Yu Hoshino
Zhiyang Zeng
Michelle Lee
Adam Weisman
Dr. Yusuke Yonamine
Dr. Keiichi Yoshimatsu
Ben Lesel
Dr. Jack Beierle
Huafang Wang
“Echo” Zhang
Walter Haberacker
Jennifer Pitzen
Prof. Pierre Baldi
Tokyo Institute of Technology
Dr.Takashi Kodama
Prof. Yoshio Okahata
University of Shizuoka
Takeo Urakami
Hiroyuki Koide
Prof. Naoto Oku
Support
NIH
Initium (ULVAC, Inc.)(QCM)
Aspira Biosystems
Shiseido
Amgen
Morphotek
JCS ( Y. H. Postdoctoral Fellowship)
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