Reporter: Liu Hongchen Advisor: Prof . Y. Sun Date: 2012.8.11

1. Preparation of High Affinity Artificial Nanoparticles Antibody and the Study of Detoxification Reporter: Liu Hongchen Advisor: Prof. Y. Sun Date: 2012.8.11

2. Background Ⅰ Ⅶ Challenges Ⅱ Target Catalog Ⅲ Plan Ⅳ Innovation Ⅴ

3. Background: Plastic antibodies • Plastic antibodies , protein -size synthetic polymer nanoparticles that are capable of recognizing and neutralizing specific biomacromoleculeswith 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.

4. 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 guanidiniumgroups can recognize a specific target protein .

5. 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 NIPAm-based 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 monodisperseand that the NPs have hydrodynamic diameters of approximately 80–100 nm in water. • Superstructure of NIPAm-based copolymers NPs.

6. Background: melittin • The toxin , melittin—a twenty-six amino acid peptide isolated from bee venom. • Melittin is a representative of membrane damaging toxins , a number of which function as key virulence factors of infectious diseases . • 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. Hydrophobic residues Positive charged residues

7. 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 hydrophobicinteractions that enable melittin to be captured by polymer NPs with high efficiency.

8. Challenge • 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. • 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 (109M-1 &1011M-1) and a narrower affinity distribution. However, the yield of high affinity part icles isolated by these strategies is quite low. Strategy 1 Strategy 2

9. 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).

10. Plan: outline Ligand density; Binding site. Preparation of NIPAm-based copolymers Adsorption equilibrium isotherm； Absorbance change. Modify the NPs with peptide ligand Affinity of modified/non-modified NPs for lysozyme Neutralization in vitro(red blood cells）； Neutralization in vivo（mice） Affinity ofmodified/non-modified NPs for melittin

11. Plan: preparation of NIPAm-based copolymers

12. Plan: modify the NPs with peptide ligand • 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. pH 4.5-7.5

13. 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.

14. Plan: Affinity of NPs for lysozyme • 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. lysodeikticusas 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.

15. Plan: Affinity of NPs for melittin • Hemolytic activity neutralization assay: • 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. A100% A0% Asample

16. Plan: Affinity of NPs for melittin • 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: Melittinwere injected into BALB/c mice slowly via tail vein. Then, NP were injected slowly via tail vein 20±5s after injection of the melittinsolution. • 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 Fluorescein-NPs Toxin Nanoparticle antidotes Blood stream

17. 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.

18. 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,etal.Design of Synthetic Polymer Nanoparticles that Capture and Neutralize a Toxic Peptide [J].Small 2009,5,No. 13, 1562-1568 . [4]ZhiyangZeng, 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. 199-204. [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.

19. Thank You!