Download PowerPoint ****

Preparation of High Affinity Artificial
Nanoparticles Antibody and the Study of
Detoxification
Reporter: Liu Hongchen
Advisor: Prof. Y. Sun
Date: 2012.8.11
Catalog
Ⅰ
Background
Ⅱ
Challenges
Ⅲ
Target
Ⅳ
Plan
Ⅴ
Ⅶ
Innovation
Background: Plastic antibodies
 Plastic antibodies , protein -size
synthetic polymer nanoparticles that are
capable of recognizing and neutralizing
specific biomacromolecules with
effectiveness comparable to antibodies ,
are of significant interest as an abiotic
alternative to antibodies .
 Adventage : a) Stable b)Low cost c)Safe
 Plastic can be used as toxin antidote,
amyloid β -peptide inhibitor et al.
Background: Plastic antibodies
 The two traditional action ways of
nanoparticles: a)Ligands covalently
attached to the surface of NPs; b) Drug
molecules encapsulated into NPs.
 Synthetic linear polymers incorporating
monomers that are capable of
recognizing hydrophobic patches and
guanidinium groups can recognize a
specific target protein .
Background: P(NIPAm-co-TBAm-AAc)
 N-isopropylacrylamide (NIPAm)
copolymers are one of the most
popular mediums for drug carrier,
inhibitor, biosensor.
 This method to prepare NIPAmbased copolymers containing 2%
cross-linker (BIS) , and acrylic acid
(AAc; negatively charged monomer),
and N- tert –butylacrylamide (TBAm;
hydrophobic monomer) .
 The solutions of NPs were
monodisperse and that the NPs have
hydrodynamic diameters of
approximately 80–100 nm in water.
 Superstructure of NIPAm-based
copolymers NPs.
Background: melittin
 The toxin , melittin—a twenty-six
amino acid peptide isolated from bee
venom.
Hydrophobic residues
 Melittin is a representative of
membrane damaging toxins , a
number of which function as key
virulence factors of infectious
diseases .
Positive charged residues  The sequence is amphiphilic, since six
amino acids at the C-terminus of the
peptide are hydrophilic, while the
remaining amino acids have a high
proportion of apolar residues.
Background: mechanism
 The affinity sites are able to interact
with melittin by both electrostatic
and hydrophobic interactions
 This copolymer is able to interact
with melittin by both electrostatic
and hydrophobic interactions that
enable melittin to be captured by
polymer NPs with high efficiency.
Challenge
Strategy 1
 The average binding affinity of the particles
to melittin ( 106 M-1) is still much weaker
than that of natural antibodies (109 M-1)
 To isolate nanoparticles with higher affinity
and a narrower affinity distribution,
nanoparticles were sorted on the basis of
peptide affinity just as antibodies , using an
affinity chromatography strategy.
Strategy 2
 To enhance the average affinity (and
perhaps increase their selectivity) for
melittin, in the final refinement,
nanoparticles are synthesized in the
presence of the target peptide melittin (the
molecular imprinting step).
 These two strategies showed much
stronger affinity (109 M-1 & 1011 M-1) and a
narrower affinity distribution. However, the
yield of high affinity part icles isolated by
these strategies is quite low.
Target
 Improve the affinity of copolymer
NPs by replacing the AAc negatively
charged monomer with stronger
negatively charged groups.
 Modify the copolymer NPs with
peptide ligand which is high
negatively charged (e.g.
tetrapeptides:DEDE).
Plan: outline
Preparation of NIPAm-based copolymers
Ligand density;
Binding site.
Modify the NPs with peptide ligand
Adsorption
equilibrium
isotherm；
Absorbance
change.
Affinity of modified/non-modified NPs for
lysozyme
Affinity of modified/non-modified NPs for
melittin
Neutralization
in vitro(red
blood cells）；
Neutralization
in vivo（mice）
Plan: preparation of NIPAm-based copolymers
Step1
Pretreatment of monomers
•NIPAm : AAc : TBAm : BIS = 48 : 20 : 40 : 2 (mol%) , TBAm (40 mol%) was
dissolved in ethanol (1 mL) before addition to the monomer solution, which
resulted in a total monomers concentration of 6.5 mM.
Step2
Degas
• The resulting solutions were degassed in a sonication bath under vacuum for 10
min and then nitrogen was bubbled through the reaction mixtures for 30 min.
Step3
Initiate the reaction
• Following the addition of ammonium persulfate aqueous solution (2.63mM)
and N,N,N’,N’-tetramethylethylenediamine (2.00mM), the polymerization was
carried out at 23–25℃ for 15–20h under a nitrogen atmosphere.
Step4
Purify
• The polymerized solutions were purified by dialysis against an excess amount
of pure water (changed more than twice a day) for >4 days.
Plan: modify the NPs with peptide ligand
pH 4.5-7.5
 EDC-mediated amide bond formation
can be used to immobilization of
ligands onto copolymer NPs. EDC
reacts with the carboxylate group of
NPs, then the activated ester
intermediate reacts with the amine
group of peptide ligand.
 Pre-experiments: Influence of
carbodiimide (EDC) to NIPAm-based
copolymer NPs.
Plan: Affinity of NPs for lysozyme
 Lysozyme, a 14 kDa basic protein
(isoelectric point (pI) = 9.3) is chosen
as target protein.
 Under the conditions of the
experiment (pH 7.3), lysozyme is
negatively charged.
 Lysozyme is cheaper compared to
melittin.
 It have reported that Lysozyme can
be captured by NPs, and both the
hydrophobic and negatively charged
groups in the NP contribute to
lysozyme capture.
Plan: Affinity of NPs for lysozyme
Incubate
Filter
Activity assay
 Add modified/non-modified NPs into
lysozyme solution(0.03mg/ml). The
resulting suspension is incubated at
37℃ for 30 min. Filter the samples.
 Lysozyme activity was assayed using
M. lysodeikticus as the substrate
dispersed in 60 mmol/L sodium
phosphate buffer (pH 6.2). In the
assay, 1.5 mL of 0.25 mg/mL
substrate solution was mixed
completely with 0.1 mL filtrate at
25◦C. The decrease in the
absorbance of the mixed solution at
450 nm was then recorded.
Plan: Affinity of NPs for melittin
 Hemolytic activity neutralization assay:
A100% A0%
Asample
 Add the melittin/NPs mixture to red
blood cell (RBC). The resulting suspension
is incubated at 37℃ for 30 min.
Centrifuge the samples. Release of the
hemoglobin is monitored by measuring
the absorbance(A sample) of the
supernatant at 415nm.
 Controls for 0 and 100% neutralization of
hemolytic activity consisted of RBCs
incubated with 1.8m M melittin without
NPs( A 0%) and a RBC suspension without
melittin and NPs (A 100%),respectively.
The percentage of neutralization was
calculated according to the left Eqation.
Plan: Affinity of NPs for melittin
Toxin
Blood stream
Nanoparticle antidotes
 Biocompatibility of NPs in Vivo: To
examine in vivo toxicity, body mass of the
mice was monitored and the sections of
kidney and liver tissues harvested from
the mice 2 weeks after injection were
examined by a pathologist as described.
 In Vivo Neutralization Assay: Melittin
were injected into BALB/c mice slowly via
tail vein. Then, NP were injected slowly
via tail vein 20±5s after injection of the
melittin solution.
 In Vivo and Ex Vivo Fluorescent Imaging
of fluorescent-labeled Melittin
 In Vivo Distribution Study of 14C Labeled
NPs
 Confocal Microscopy Imaging and
Analysis of Cy5-melittin and FluoresceinNPs
Innovation
 Modify the high affinity peptides ligand on the surface
of copolymer nanoparticles, to improve the copolymer
nanoparticles binding capacity to target protein.
 If the experiment is successful, we will get a new
nanoparticle, which can be used as toxin antidote,
abeta & P53 inhibitor.
References
[1] Keiichi Yoshimatsu, Benjamin K. Lesel, et al, Tenperature- Responsive “Catch and Release”
of Proteins by using Multifunctional Polymer-Based Nanoparticles [J], Angew. Chem. Int. Ed.
2012,51, 2405- 2408.
[2] Yu Hoshino, Walter W,et al, Affinity Purification of Multifunctional Polymer [J], J. AM. CHEM.
SOC. 2010, 132, 13648-13650.
[3]Yu Hoshino, Takeo Urakami, Takashi Kodama,et al.Design of Synthetic Polymer
Nanoparticles that Capture and Neutralize a Toxic Peptide [J].Small 2009,5,No. 13, 15621568 .
[4]Zhiyang Zeng,Yu Hoshino, Andy Rodriguez, et al.Synthetic Polymer Nanoparticles with
Antibody-like Affinity for a Hydrophilic Peptide [J]. ACS NANO. 2010.VOL.4. No.1. 199204.
[5]Yu Hoshino, Kenneth J. Shea.The evolution of plastic antibodies [J]. J. Master. Chem., 2011,
21, 3517-3521.
[6]Yu Hoshino, Hiroyuki Koide, et al. The rational design of a synthetic polymer nanoparticle
that neutralizes a toxic peptide in vivo[J]. PNAS. January 3, 2012.Vol.109. No.1. 33-38.
[7] Robb M J, Connal L A, Lee B F, et al. Polym. Chem. 2012, 3: 1618.
[8] Shan J, Tenhu H Chem. Commun. 2007: 4580.
[9] Xu P,Van Kirk E A, Li S, et al. Colloids and Surfaces B: Biointerfaces 2006, 48: 50.
[10] Zubarev E R, Xu J, Sayyad A, et al. Journal of the American Chemical Society 2006, 128:
15098.
[11] Ulbrich K, Michaelis M, Rothweiler F, et al. International Journal of Pharmaceutics 2011,
406: 128.
Thank You!