Synthetic polymer nanoparticles (NPs) that bind venomous molecules and neutralize their function in vivo are of significant interest as "plastic antidotes." Recently, procedures to synthesize polymer NPs with affinity for target peptides have been reported. However, the performance of synthetic materials in vivo is a far greater challenge. Particle size, surface charge, and hydrophobicity affect not only the binding affinity and capacity to the target toxin but also the toxicity of NPs and the creation of a "corona" of proteins around NPs that can alter and or suppress the intended performance. Here, we report the design rationale of a plastic antidote for in vivo applications. Optimizing the choice and ratio of functional monomers incorporated in the NP maximized the binding affinity and capacity toward a target peptide. Biocompatibility tests of the NPs in vitro and in vivo revealed the importance of tuning surface charge and hydrophobicity to minimize NP toxicity and prevent aggregation induced by nonspecific interactions with plasma proteins. The toxin neutralization capacity of NPs in vivo showed a strong correlation with binding affinity and capacity in vitro. Furthermore, in vivo imaging experiments established the NPs accelerate clearance of the toxic peptide and eventually accumulate in macrophages in the liver. These results provide a platform to design plastic antidotes and reveal the potential and possible limitations of using synthetic polymer nanoparticles as plastic antidotes.

We report that multi functional 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 thermo-responsiveness of the nanoparticles allowing "catch and release" of the target peptide by adjusting the temperature. Purified particles show much stronger affinity (Kd app ≈ nM) and a narrower affinity distribution than the average of particles before purification (Kd app > μM) in room temperature, but can release the peptide just by changing 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.General procedures for the creation of synthetic materials with biomacromolecular recognition sites are of significant interest as a route to stable, robust and mass-produced substitutes for antibodies. [1][2][3][4][5][6][7][8] Ideally, recognition of complex biological targets, including proteins, peptides and carbohydrates, requires multiple functional groups that contact target molecules by a combination of electrostatic, 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 polymer materials with molecular recognition sites. [1][2][3][4][5] However, in contrast to antibodies whose exact sequence can be determined and cloned, polymerized materials result in heterogeneous structures with a distribution of recognition sites. 1,3,7 This is an intrinsic property of polymers synthesized under kinetic control, in contrast to the synthetic small molecular hosts prepared by multi step reactions 9 or by self-assembly under equilibrating conditions 10 . 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 multi-functional copolymer nanoparticles (MFNPs). These nanoparticles are approximately the size of a large protein and are "purified" on the basis of peptide affinity just as antibodies, using an affinity chromatography strategy. The concept of affinity purification of NPs was demonstrated with melittin, a 26 amino acid peptide ( fig. 1a), as the target molecule. Melittin has six positive charges of which four are localized in a hydrophilic six amino acid sequence on the C-terminus. The remaining twenty amino acids have a high proportion of apolar residues. 11For the MFNPs, we chose cross-linked N-isopropylacrylamide NPs (~30 nm) incorporating hydrophobic (N-t-butylacrylamide (TBAm)) and negatively charged (acrylic acid (AAc)) functional monomers ( fig. 1b). We have reported that NPs with this composition interacts with melittin (K dapp = 46 μM) via both electrostatic-and hydrophobi nteractions in PBS (35mM phosphate buffer/0.15 M NaCl, pH 7.3). 5 However, th...

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