Abstract
Synthetic polymer nanoparticles (NPs) with intrinsic affinity for target biomacromolecules hold great promise in the development of novel tools for biological and biomedical research. We recently reported the design and synthesis of abiotic, synthetic polymer NPs with high intrinsic affinity for a peptide toxin melittin. The NP was selected by screening a small library of NPs (ā¼100 nm) composed of various ratios of monomers that contain functional groups complementary to the peptide melittin. The selected polymer NP, a co-polymer of acrylic acid (AAc), N-tert-butylacrylamide (TBAm), N-isopropylacrylamide (NIPAm) and N,Nā²-methylenebisacrylamide (BIS), effectively captures and neutralizes the toxicity of the peptide through a combination of electrostatic and hydrophobic interactions. This protocol describes a step-by-step procedure for the preparation and evaluation of synthetic polymer NPs for sequestration and neutralization of the target peptide toxin. The polymer NPs can be synthesized in a one-step polymerization reaction using commercially available reagents. The polymerization reaction for the synthesis of polymer NPs takes several hours, and the total protocol including subsequent purification and characterization by dynamic light scattering, NMR and toxicity neutralization assays takes 1ā2 weeks in total.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kasturiratne, A. et al. The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med. 5, 1591ā1604 (2008).
Gutierrez, J., Williams, D., Fan, H. & Warrell, D. Snakebite envenoming from a global perspective: towards an integrated approach. Toxicon 56, 1223ā1235 (2010).
Rainey, G. & Young, J. Antitoxins: novel strategies to target agents of bioterrorism. Nat. Rev. Microbiol. 2, 721ā726 (2004).
Espino-Solis, G., Riano-Umbarila, L., Becerril, B. & Possani, L. Antidotes against venomous animals: state of the art and prospectives. J. Proteomics 72, 183ā199 (2009).
Spiller, H., Bosse, G. & Ryan, M. Use of antivenom for snakebites reported to United States poison centers. Am. J. Emerg. Med. 28, 780ā785 (2010).
Simpson, I. Time for an alternative perspective: the eternal problem of supply and quality of anti snake venom in the developing worldā'It's the economy, stupid'. Wilderness Environ. Med. 19, 186ā194 (2008).
Hoshino, Y., Kodama, T., Okahata, Y. & Shea, K.J. Peptide imprinted polymer nanoparticles: a plastic antibody. J. Am. Chem. Soc. 130, 15242ā15243 (2008).
Hoshino, Y. et al. Design of synthetic polymer nanoparticles that capture and neutralize a toxic peptide. Small 5, 1562ā1568 (2009).
Hoshino, Y. et al. The rational design of a synthetic polymer nanoparticle that neutralizes a toxic peptide in vivo. Proc. Natl. Acad. Sci. USA 109, 33ā38 (2012).
Hu, C., Fang, R., Copp, J., Luk, B. & Zhang, L. A biomimetic nanosponge that absorbs pore-forming toxins. Nat. Nanotechnol. 8, 336ā340 (2013).
Weisman, A., Chen, Y.A., Hoshino, Y., Zhang, H. & Shea, K.J. Engineering nanoparticle antitoxins utilizing aromatic interactions. Biomacromolecules 15, 3290ā3295 (2014).
Yoshimatsu, K. et al. Epitope discovery for a synthetic polymer nanoparticle: a new strategy for developing a peptide tag. J. Am. Chem. Soc. 136, 1194ā1197 (2014).
Mahon, C.S. & Fulton, D.A. Mimicking nature with synthetic macromolecules capable of recognition. Nat. Chem. 6, 665ā672 (2014).
Hoshino, Y., Lee, H. & Miura, Y. Interaction between synthetic particles and biomacromolecules: fundamental study of nonspecific interaction and design of nanoparticles that recognize target molecules. Polym. J. 46, 537ā545 (2014).
Pelton, R. & Chibante, P. Preparation of aqueous lattices with N-isopropylacylamide. Colloids Surf. 20, 247ā256 (1986).
Pelton, R. Temperature-sensitive aqueous microgels. Adv. Colloid Interface Sci. 85, 1ā33 (2000).
Mcphee, W., Tam, K. & Pelton, R. Poly(N-isopropylacrylamide) lattices prepared with sodium dodecyl sulfate. J. Colloid Interface Sci. 156, 24ā30 (1993).
Wu, X., Pelton, R., Hamielec, A., Woods, D. & Mcphee, W. The kinetics of poly(N-isopropylacrylamide) microgel latex formation. Colloid Polym. Sci. 272, 467ā477 (1994).
Ito, S. et al. Preparation of thermosensitive submicrometer gel particles with anionic and cationic charges. Langmuir 15, 4289ā4294 (1999).
Debord, J.D. & Lyon, L.A. Synthesis and characterization of pH-responsive copolymer microgels with tunable volume phase transition temperatures. Langmuir 19, 7662ā7664 (2003).
Armarego, W.L.F. & Chai, C.L.L. Purification of Laboratory Chemicals 6th edn. (Butterworth Heinemann, Elsevier Inc., 2009).
Haberman, E. & Zeuner, G. Comparative studies of native and synthetic melittins. Naunyn-Schmiedeberg's Arch. 270, 1ā9 (1971).
Son, D. et al. Melittin inhibits vascular smooth muscle cell proliferation through induction of apoptosis via suppression of nuclear factor-kappa B and Akt activation and enhancement of apoptotic protein expression. J. Pharmacol. Exp. Ther. 317, 627ā634 (2006).
Li, B. et al. Growth arrest and apoptosis of the human hepatocellular carcinoma cell line Bel-7402 induced by melittin. Onkologie 29, 367ā371 (2006).
Gajski, G. & Garaj-Vrhovac, V. Melittin: a lytic peptide with anticancer properties. Environ. Toxicol. Pharmacol. 36, 697ā705 (2013).
Yoshimatsu, K. et al. Temperature-responsive ācatch and releaseā of proteins by using multifunctional polymer-based nanoparticles. Angew. Chem. Int. Ed. Engl. 51, 2405ā2408 (2012).
Lee, S.-H. Engineered synthetic polymer nanoparticles as IgG affinity ligands. J. Am. Chem. Soc. 134, 15765ā15772 (2012).
Yonamine, Y. et al. Polymer nanoparticle-protein interface. Evaluation of the contribution of positively charged functional groups to protein affinity. ACS Appl. Mater. Interfaces 5, 374ā379 (2013).
Nayak, S. & Lyon, L.A. Soft nanotechnology with soft nanoparticles. Angew. Chem. Int. Ed. Engl. 44, 7686ā7708 (2005).
Kawaguchi, H. Thermoresponsive microhydrogels: preparation, properties and applications. Polym. Int. 63, 925ā932 (2014).
Islam, M., Gao, Y., Li, X. & Serpe, M. Responsive polymers for biosensing and protein delivery. J. Mater. Chem. B 2, 2444ā2451 (2014).
Cedervall, T. et al. Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc. Natl. Acad. Sci. USA 104, 2050ā2055 (2007).
Lindman, S. et al. Systematic investigation of the thermodynamics of HSA adsorption to N-iso-propylacrylamide/N-tert-butylacrylamide copolymer nanoparticles. Effects of particle size and hydrophobicity. Nano Lett. 7, 914ā920 (2007).
Linse, S. et al. Nucleation of protein fibrillation by nanoparticles. Proc. Natl. Acad. Sci. USA 104, 8691ā8696 (2007).
Cabaleiro-Lago, C. et al. Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles. J. Am. Chem. Soc. 130, 15437ā15443 (2008).
Cabaleiro-Lago, C., Lynch, I., Dawson, K.A. & Linse, S. Inhibition of IAPP and IAPP((20-29)) fibrillation by polymeric nanoparticles. Langmuir 26, 3453ā3461 (2010).
Luchini, A. et al. Smart hydrogel particles: Biomarker harvesting: one-step affinity purification, size exclusion, and protection against degradation. Nano Lett. 8, 350ā361 (2008).
Smith, M.H. & Lyon, L.A. Tunable encapsulation of proteins within charged microgels. Macromolecules 44, 8154ā8160 (2011).
Smith, M.H. & Lyon, L.A. Multifunctional nanogels for siRNA delivery. Acc. Chem. Res. 45, 985ā993 (2012).
Yonamine, Y., Hoshino, Y. & Shea, K.J. ELISA-mimic screen for synthetic polymer nanoparticles with high affinity to target proteins. Biomacromolecules 13, 2952ā2957 (2012).
Zeng, Z. et al. Synthetic polymer nanoparticle-polysaccharide interactions: a systematic study. J. Am. Chem. Soc. 134, 2681ā2690 (2012).
Beierle, J.M. et al. Polymer nanoparticle hydrogels with autonomous affinity switching for the protection of proteins from thermal stress. Angew. Chem. Int. Ed. Engl. 53, 9275ā9279 (2014).
Meunier, F., Elaissari, A. & Pichot, C. Preparation and characterization of cationic poly(N-isopropylacrylamide) copolymer latexes. Polym. Adv. Technol. 6, 489ā496 (1995).
Hu, X., Tong, Z. & Lyon, L. Synthesis and physicochemical properties of cationic microgels based on poly(N-isopropylmethacrylamide). Colloid Polym. Sci. 289, 333ā339 (2011).
Hoshino, Y., Imamura, K., Yue, M., Inoue, G. & Miura, Y. Reversible absorption of CO2 triggered by phase transition of amine-containing micro- and nanogel particles. J. Am. Chem. Soc. 134, 18177ā18180 (2012).
Hu, X., Tong, Z. & Lyon, L. Control of poly(N-isopropylacrylamide) microgel network structure by precipitation polymerization near the lower critical solution temperature. Langmuir 27, 4142ā4148 (2011).
Candau, F., Leong, Y., Pouyet, G. & Candau, S. Inverse microemulsion polymerization of acrylamideācharacterization of the water-in-oil microemulsions and the final microlatexes. J. Colloid Interface Sci. 101, 167ā183 (1984).
Antonietti, M., Basten, R. & Lohmann, S. Polymerization in microemulsionsāa new approach to ultrafine, highly functionalized polymer dispersions. Macromol. Chem. Phys. 196, 441ā466 (1995).
Acknowledgements
This research was supported by the US National Science Foundation (DMR-1308363). H.K. is a recipient of the Japan Society for the Promotion of Science (JSPS) Fellowship.
Author information
Authors and Affiliations
Contributions
K.Y., H.K., Y.H. and K.J.S. developed protocols. K.Y. and H.K. contributed all the data. K.Y., H.K. and K.J.S. wrote the paper. All authors have discussed the results and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Table 1 (PDF 88 kb)
Supplementary Data 1
1H NMR spectrum (500 MHz, CD3OD) of polymer NP synthesized by the copolymerization of 5 mol% AAc, 40 mol% TBAm, 53 mol% NIPAm, and 2 mol% BIS (values are in monomer feed ratio). (ZIP 88 kb)
Supplementary Data 2
13C NMR spectrum (126 MHz, CD3OD) of polymer NP synthesized by the copolymerization of 5 mol% AAc, 40 mol% TBAm, 53 mol% NIPAm, and 2 mol% BIS (values are in monomer feed ratio). (ZIP 225 kb)
Rights and permissions
About this article
Cite this article
Yoshimatsu, K., Koide, H., Hoshino, Y. et al. Preparation of abiotic polymer nanoparticles for sequestration and neutralization of a target peptide toxin. Nat Protoc 10, 595ā604 (2015). https://doi.org/10.1038/nprot.2015.032
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2015.032
This article is cited by
-
Emerging functional materials based on chemically designed molecular recognition
BMC Materials (2020)
-
Evolution of macromolecular complexity in drug delivery systems
Nature Reviews Chemistry (2017)
-
A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165)
Nature Chemistry (2017)
-
Solid-phase synthesis of molecularly imprinted nanoparticles
Nature Protocols (2016)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
