Biochemical diversity of venom extracts often occurs within a small number of shared protein families. Developing a sequestrant capable of broad-spectrum neutralization across various protein isoforms within these protein families is a necessary step in creating broad-spectrum antivenom. Using directed synthetic evolution to optimize a nanoparticle (NP) formulation capable of sequestering and neutralizing venomous phospholipase A (PLA), we demonstrate that broad-spectrum neutralization and sequestration of venomous biomacromolecules is possible via a single optimized NP formulation. Furthermore, this optimized NP showed selectivity for venomous PLA over abundant serum proteins, was not cytotoxic, and showed substantially long dissociation rates from PLA. These findings suggest that it may show efficacy as an in vivo venom sequestrant and may serve as a generalized lipid-mediated toxin sequestrant.

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ABSTRACT:The specific binding and uptake of protein molecules to individual hydrogel nanoparticles is measured with real-11 time single-nanoparticle surface plasmon resonance imaging (SPRI) microscopy. Nanoparticles that adsorb onto chemically 12 modified gold thin films interact with traveling surface plasmon polaritons and create individual point diffraction patterns in the 13 SPRI microscopy differential reflectivity images. The intensity of each point diffraction pattern depends on the integrated 14 refractive index of the nanoparticle; an increase in this single nanoparticle point diffraction intensity (Δ%R NP ) is observed for 15 nanoparticles that bind proteins. SPRI adsorption measurements can be used to measure an average increase in Δ%R NP that can 16 be correlated with bulk dynamic light scattering measurements. Moreover, the distribution of Δ%R NP values observed for 17 individual nanoparticles can be used to learn more about the nature of the protein−nanoparticle interaction. As a first example, 18 the binding of the lectin Concanavalin A to 180 nm N-isopropylacrylamide hydrogel nanoparticles that incorporate a small 19 percentage of mannose sugar monomer units is characterized. ■ INTRODUCTION21 Hydrogel nanoparticles (HNPs) are unique synthetic nanoma-22 terials that can incorporate various chemical functionalities 23 specifically designed to capture and release proteins, peptides, 24 or other small molecules. These capabilities have led to a 25 significant interest in the potential use of HNPs in biomedical 26 applications such as targeted drug delivery, medical diagnostics, 27 and biosensing. 1−6 For example, NIPAm-based (N-isopropyla-28 crylamide) HNPs have been utilized for detection of various 29 biomolecules, such as DNA, 7,8 proteins, 9−11 and other 30 biologically relevant small molecules. 12,13 Additionally, the 31 specific uptake of proteins into HNPs can also be used as a 32 model system for studying various biological phenomena such 33 as multivalent lectin−carbohydrate interactions. 14−20 For all of 34 these applications, it is essential that the uptake of proteins into 35 individual nanoparticles be quantitated and analyzed. For the 36 case of fluorescently labeled proteins, single nanoparticle 37 fluorescence imaging can be used to monitor affinity uptake 38 into single HNPs. 21 67 We showed that although the average HNP size (as measured 68 by DLS) did not change with melittin concentration the 69 average Δ%R NP varied linearly due to melittin uptake into the 70 HNPs. 71In this paper, we extend our use of single-nanoparticle SPRI 72 microscopy to monitor the specific adsorption and uptake of 73 proteins to individual HNPs. We have synthesized NIPAm-74 based HNPs that incorporate a small percentage of monomers 75 modified with mannose sugar units into the hydrogel polymer 76 as shown in Figure 1b. We then used SPRI microscopy to 77 monitor the interactions of the lectin Concanavalin A (Con A) 78 to these mannose-incorporated HNPs (mHNPs), shown in 79 Figure 1c. Bot...

The mechanism of amyloidosis of amyloid beta (1-42) (Abeta (1-42)) was investigated by the well-defined glycocluster interface. We prepared monovalent, divalent, and trivalent 6-sulfo-N-acetyl-d-glucosamine (6S-GlcNAc) immobilized substrates. The morphology and secondary structure of Abeta (1-42) aggregates on the substrates were investigated by dynamic-mode AFM and FTIR-RAS. Abeta (1-42) interactions with multivalent sugars were evaluated by surface plasmon resonance, and the cytotoxicity of Abeta (1-42) to HeLa cells was evaluated by MTT assay. Morphological images showed, interestingly, that Abeta (1-42) aggregates had a tendency to form globules rather than fibrils as the valency of 6S-GlcNAc on the substrate was increased. The SPR measurements indicated that this morphological change of Abeta (1-42) was related to the change of binding mode, and the binding mode was dependent on the multivalency of the sugar. Globular Abeta (1-42) was more toxic than fibrillar Abeta (1-42) to HeLa cells. These results suggested that the multivalency of sugars for the amyloidosis of Abeta (1-42) was significant in its morphology and aggregation effects at the surface of the cell membrane mimic.

We describe an approach for the discovery of protein affinity reagents (PARs). Abiotic synthetic hydrogel copolymers can be "tuned" for selective protein capture by the type and ratios of functional monomers included in their polymerization and by the polymerization conditions (i.e., pH). By screening libraries of hydrogel nanoparticles (NPs) containing charged and hydrophobic groups against a protein target (IgG), a stimuliresponsive PAR is selected. The robust carbon backbone synthetic copolymer is rapidly synthesized in the chemistry laboratory from readily available monomers. The production of the PAR does not require living cells and is free from biological contamination. The capture and release of the protein by the copolymer NP is reversible. IgG is sequestered from human serum at pH 6.5 and following a wash step, the purified protein is released by elevating the pH to 7.3. The binding and release of the protein occur without denaturation. The abiotic material functions as a selective PAR for the F(ab′) 2 domain of IgG for pull-down and immunoprecipitation experiments and for isolation and purification of proteins from complex biological mixtures.