Frequently Asked Questions
Synthetic polymer nanomaterials capable of interacting with target biomacromolecules with high affinity and specificity have been developed for the detection of target proteins, nucleic acids, and small molecules for diagnostics, and for controlling the on and off state of enzyme activity. In addition, synthetic copolymers have been developed for cancer therapy, and for tissue engineering by mimicking protein-protein interactions. Synthetic linear polymers, dendrimers, and NPs are attractive candidates as cost-effective protein affinity reagent alternatives to antibodies. Within this group, functionalized hydrogel NPs represent a particularly attractive biocompatible, functional material. For example, the affinity of nontoxic hydrogel NPs against target biomacromolecules can be engineered by incorporating combinations of hydrophobic and charged groups in a process described as directed chemical evolution. Optimized NPs have exhibited nanomolar affinity against target biomacromolecules and have demonstrated utility by neutralizing biological function in
vitro and/or in vivo.
Synthetic antibodies often function with affinity and selectivity for proteins and peptides (antigens) that is comparable to traditional biological antibodies. However, synthetic antibodies are fundamentally different materials from protein antibodies. They are synthesized by free radical polymerization, a kinetic process that produces statistical carbon backbone copolymers, low information content materials that lack the sequence specificity of biological antibodies. Both synthetic and biological antibodies function by "recognizing" and binding to protein and peptide antigens.
HOW DO THE BINDING CHARACTERISTICS OF SYNTHETIC ANTIBODIES DIFFER FROM THOSE OF NATURAL ANTIBODIES?
At the molecular level, biological antibodies present hydrophobic, electrostatic and hydrogen bonding groups to a complementary surface on the antigen. The surface contacts can span 1500 sq. Å and involve 20-25 amino acid close contacts. The complementary information is contained in the amino acid sequence of the protein antibody which is ultimately traced back to the protein template. Precise (atomistic) understanding of the "recognition" can be obtained from X-ray crystallography. The process is understood as deterministic. Synthetic antibodies on the other hand are not pure substances, their properties are determined as averages of the ensemble. The 3D structure of the hydrogel nanoparticle might be described as a 3- dimensional spider web with the voids between the polymer strands comprised of solvent and solute. The carbon backbone strands are highly flexible and dynamic. The interaction with the antigen can occur along linear sequences or discontinuous sequences or and between folds and cross links and linear sequences. Given the size and statistical nature of the sequences, efforts to understand binding by deterministic means, is not possible, and sequences within this polymer are not available by any existing technology.
GIVEN THEIR STATISTICAL NATURE, HOW ARE HYDROGEL NPS ENGINEERED TO BIND TO A SPECIFIC TARGET WITH SIMULTANEOUSLY HIGH AFFINITY AND HIGH SELECTIVITY?
Hydrogel copolymers are statistical copolymers incorporating combinations and permutations of functional monomers that include N-tert- butylacrylamide (TBAm, hydrophobic monomer), acrylic acid (AAc, negatively charged monomer), N-(3-aminopropyl)-2-methylacrylamide hydrochloride (APM, positively charged monomer), and/or 3,4,6-sulfo-N-acetylglucosamine (GNA), and others. Monomer sequences in the quaternary copolymer are determined by the relative reactivity ratios of the monomers and their instantaneous concentration. The binding profile of the resulting NP can be tuned by adjusting these reactivity ratios.
For example, the ratios (40% TBAm, 20% AAc, 2% BIS, 38% NIPAm) produce NPs which bind with high affinity and selectivity to histone, a toxic cationic protein associated with sepsis, while the ratios (40% TBAm, 2% GNA, 56% NIPAm) produce NPs which bind to VEGF, a protein implicated in macular degeneration, with high affinity and selectivity.
In engineering NPs, we draw upon our past experience in choosing the type of polymer backbone, functionality and composition to improve our chances of identifying polymers with high affinity to a target. Even still, discovery has an empirical component. What we can do to compensate is assemble a very large number of combinations and permutations of selected functional monomers in, for example, a high MW hydrogel NP, and screen the ensemble of NPs for a "hit". By synthesizing the polymer ensemble reproducibly with the same function, we can conclude that the polymer has a high probability of binding selectively.
WHAT IS THE PHYSICAL STRUCTURE OF A HYDROGEL COPOLYMER?
For the sake of concreteness, consider a hydrogel copolymer with high binding affinity to histone engineered with reactivity ratios of (40% TBAm, 20% AAc, 2% BIS, 38% NIPAm). The average monomer molecular weight of these copolymers is ~111 g/mole. The average MW of each NP is ~ 50 million, and each copolymer contains approximately 450,000 monomers. The polymers have densities approximately 1/30th to 1/100th that of a globular protein. They are mostly water. The average mesh size of ~10 nm is large enough to accommodate peptides and most proteins throughout their interior. As detailed above, the 3D structure of the hydrogel nanoparticle can be described as a 3- dimensional spider web with the voids between the polymer strands comprised of solvent and solute.