Engineering the Protein Corona of a Synthetic Polymer Nanoparticle for Broad-Spectrum Sequestration and Neutralization of Venomous Biomacromolecules

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 A₂ (PLA₂), 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 PLA₂ over abundant serum proteins, was not cytotoxic, and showed substantially long dissociation rates from PLA₂. These findings suggest that it may show efficacy as an in vivo venom sequestrant and may serve as a generalized lipid-mediated toxin sequestrant

Polymer Nanoparticle–Protein Interface. Evaluation of the Contribution of Positively Charged Functional Groups to Protein Affinity

Cationic-functionalized polymer nanoparticles (NPs) show strikingly distinct affinities to proteins depending on the nature of the cationic functional group. <i>N</i>-Isopropylacrylamide (NIPAm) polymer NPs incorporating three types of positively charged functional groups (guanidinium, primary amino, and quaternary ammonium groups) were prepared by precipitation polymerization. The affinities to fibrinogen, a protein with an isoelectric point (pI) of 5.5, were compared using UV–vis spectrometry and a quartz crystal microbalance (QCM). Guanidinium-containing NPs showed the highest affinity to fibrinogen. The observation is attributed to strong, specific interactions with carboxylate groups on the protein surface. The affinity of the positively charged NPs to proteins with a range of pIs revealed that protein-NP affinity is due to a combination of ionic, hydrogen bonding, and hydrophobic interactions. Protein affinity can be modulated by varying the composition of these functional monomers in the acrylamide NPs. Engineered NPs containing the guanidinium group with hydrophobic and hydrogen bonding functional groups were used in an affinity precipitation for the selective separation of fibrinogen from a plasma protein mixture. Circular dichroism (CD) revealed that the protein was not denatured in the process of binding or release

Light-Triggered Charge Reversal of Organic–Silica Hybrid Nanoparticles

A functional nanoparticle with light-triggered charge reversal based on a protected amine-bridged polysilsesquioxane was designed. An emulsion- and amine-free sol–gel synthesis was developed to prepare uniform nanospheres. Photolysis of suspensions of these nanoparticles results in a reversal of the ζ potential. This behavior has been used to trigger nanoparticle self-assembly, nanocomposite hydrogel formation, and nanoparticle release, showing the potential of this material in nanoscale manipulation and nanoparticle therapy

Synthetic Polymer Nanoparticle–Polysaccharide Interactions: A Systematic Study

The interaction between synthetic polymer nanoparticles (NPs) and biomacromolecules (e.g., proteins, lipids, and polysaccharides) can profoundly influence the NPs fate and function. Polysaccharides (e.g., heparin/heparin sulfate) are a key component of cell surfaces and the extracelluar matrix and play critical roles in many biological processes. We report a systematic investigation of the interaction between synthetic polymer nanoparticles and polysaccharides by ITC, SPR, and an anticoagulant assay to provide guidelines to engineer nanoparticles for biomedical applications. The interaction between acrylamide nanoparticles (∼30 nm) and heparin is mainly enthalpy driven with submicromolar affinity. Hydrogen bonding, ionic interactions, and dehydration of polar groups are identified to be key contributions to the affinity. It has been found that high charge density and cross-linking of the NP can contribute to high affinity. The affinity and binding capacity of heparin can be significantly diminished by an increase in salt concentration while only slightly decreased with an increase of temperature. A striking difference in binding thermodynamics has been observed when the main component of a polymer nanoparticle is changed from acrylamide (enthalpy driven) to <i>N</i>-isopropylacryalmide (entropy driven). This change in thermodynamics leads to different responses of these two types of polymer NPs to salt concentration and temperature. Select synthetic polymer nanoparticles have also been shown to inhibit protein–heparin interactions and thus offer the potential for therapeutic applications

Engineered Synthetic Polymer Nanoparticles as IgG Affinity Ligands

A process for the preparation of an abiotic protein affinity ligand is described. The affinity ligand, a synthetic polymer hydrogel nanoparticle (NP), is formulated with functional groups complementary to the surface presentation of the target protein. An iterative process is used to improve affinity by optimizing the composition and proportion of functional monomers. Since the polymer NPs are formed by a kinetically driven process, the sequence of functional monomers in the polymer chain is not controlled; only the average composition can be adjusted by the stoichiometry of the monomers in the feed. To compensate for this the hydrogel NP is lightly cross-linked resulting in chain flexibility that takes place on a submillisecond time scale allowing the polymer to “map” onto a protein surface with complementary functionality. In this study, we report a lightly cross-linked (2%) <i>N</i>-isopropyl acrylamide (NIPAm) synthetic polymer NP (50–65 nm) incorporating hydrophobic and carboxylate groups that binds with high affinity to the Fc fragment of IgG. The affinity and amount of NP bound to IgG is pH dependent. The hydrogel NP inhibits protein A binding to the Fc domain at pH 5.5, but not at pH 7.3. A computational analysis was used to identify potential NP–protein interaction sites. Candidates include a NP binding domain that overlaps with the protein A–Fc binding domain at pH 5.5. The computational analysis supports the inhibition experimental results and is attributed to the difference in the charged state of histidine residues. Affinity of the NP (3.5–8.5 nM) to the Fc domain at pH 5.5 is comparable to protein A at pH 7. These results establish that engineered synthetic polymer NPs can be formulated with an intrinsic affinity to a specific domain of a large biomacromolecule

Biomimetic Design of Mussel-Derived Bioactive Peptides for Dual-Functionalization of Titanium-Based Biomaterials

Specific cell adhesion and osteogenicity are both crucial factors for the long-term success of titanium implants. In this work, two mussel-derived bioactive peptides were designed to one-step dual-biofunctionalization of titanium implants via robust catechol/TiO₂ coordinative interactions. The highly biomimetic peptides capped with integrin-targeted sequence or osteogenic growth sequence could efficiently improve the biocompatibilities of titanium implants and endow the implants with abilities to induce specific cell adhesion and enhanced osteogenicity. More importantly, rationally combined use of the two biomimetic peptides indicated an enhanced synergism on osteogenicity, osseointegration and finally the mechanical stability of Ti implants in vivo. Therefore, the highly biomimetic mussel-derived peptides and the dual-functional strategy in this study would provide a facile, safe, and effective means for improving clinical outcome of titanium-based medical implants

Synthetic Polymer Affinity Ligand for <i>Bacillus thuringiensis</i> (<i>Bt</i>) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the <i>Bt</i> Protein–Insect Receptor Binding Mechanism

We report a novel strategy for creating abiotic Bacillus thuringiensis (<i>Bt</i>) protein affinity ligands by biomimicry of the recognition process that takes place between <i>Bt</i> Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R₁ proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes ²⁸⁰FRGS­AQGI­EGS²⁹⁰, ³⁶⁸RRPF­NIGI­NNQQ³⁷⁹ and ⁴³⁶FRSG­FSNS­SVSIIR⁴⁴⁹ located in loop α8, loop 2 and loop 3 of domain II of <i>Bt</i> Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, ³⁶⁸RRPF­NIGIN­NQQ³⁷⁹. This same NP also had specific binding ability to both <i>Bt</i> Cry1Ab and <i>Bt</i> Cry1Ac, proteins that share the same epitope, but very low affinity to <i>Bt</i> Cry2A, <i>Bt</i> Cry1C and <i>Bt</i> Cry1F closely related proteins that lack epitope homology. To locate possible NP-<i>Bt</i> Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of ⁸⁶⁵NITI­HITD­TNNK⁸⁷⁶, a specific recognition site in insect receptor Bt-R₁, to ³⁶⁸RRPF­NIGIN­NQQ³⁷⁹. The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to <i>Bt</i> Cry1Ab/Ac proteins mainly via ³⁶⁸RRPF­NIGIN­NQQ³⁷⁹. This epitope region was then utilized as a “target” or “bait” for the separation and concentration of <i>Bt</i> Cry1Ac protein from the extract of transgenic <i>Bt</i> cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands