High-Affinity Points of Interaction on Antibody Allow Synthesis of Stable and Highly Functional Antibody–Gold Nanoparticle Conjugates

Abstract

Many emerging nanobiotechnologies rely on the proper function of proteins immobilized on gold nanoparticles. Often, the surface chemistry of the AuNP is engineered to control the orientation, surface coverage, and structure of the adsorbed protein to maximize conjugate function. Here, we chemically modified antibody to investigate the effect of protein surface chemistries on adsorption to AuNPs. A monoclonal anti-horseradish peroxidase IgG antibody (anti-HRP) was reacted with N-succinimidyl acrylate (NSA) or reduced dithiobissuccinimidyl propionate (DSP) to modify lysine residues. Zeta potential measurements confirmed that both chemical modifications reduced the localized regions of positive charge on the protein surface, while the DSP modification incorporated additional free thiols. Dynamic light scattering confirmed that native and chemically modified antibodies adsorbed onto AuNPs to form bioconjugates; however, adsorption kinetics revealed that the NSA-modified antibody required significantly more time to allow for the formation of a hard corona. Moreover, conjugates formed with the NSA-modified antibody lost antigen-binding function, whereas unmodified and DSP-modified antibodies adsorbed onto AuNPs to form functional conjugates. These results indicate that high-affinity functional groups are required to prevent protein unfolding and loss of function when adsorbed on the AuNP surface. The reduced protein charge and high-affinity thiol groups on the DSP-modified antibody enabled pH-dependent control of protein orientation and the formation of highly active conjugates at solution pHs (<7.5) that are inaccessible with unmodified antibody due to conjugate aggregation. This study establishes parameters for protein modification to facilitate the formation of highly functional and stable protein–AuNP conjugates.

Simultaneous Stabilization and Functionalization of Gold Nanoparticles via Biomolecule Conjugation: Progress and Perspectives

“…Antibodies can be attached to AuNP surfaces via chemisorption, electrostatic or hydrophobic interactions, and bonding to target functionalities such as cysteine and amine groups. − Currently, the covalent bioconjugation of antibodies is preferred because it most effectively stabilizes AuNPs compared with other functionalization methods. Two major covalent bioconjugation methods exist for AuNPs: (1) direct conjugation between thiol group-containing antibodies and AuNPs , and (2) indirect conjugation between the amide groups of antibodies and PEGylated carboxyl groups around AuNPs . The direct conjugation of antibodies via Au–thiol bonding is a simple and fast method for AuNP functionalization; however, it faces the critical issues of nonspecific interactions and unwanted protein adsorption in biological media.…”

Antibody‐Incorporated Nanomedicines for Cancer Therapy

“…Another thiolation reagent 2-IT has also been applied in different studies for antibody modification. [25,[146][147][148][149] Trabulo et al modified iron oxide MNPs with the chemotherapeutic drug gemcitabine (Gem) and anti-CD47 antibody both via disulfide bonds, and the resultant MNPs achieved targeted killing of CD47-positive pancreatic cancer cells. [25] As shown in Figure 5d, the thiol groups on MNPs were introduced by the reaction between cysteamine and the carboxyl groups of dimercaptosuccinic acid-coated MNPs through EDC/NHS chemistry.…”

“…[ 93 ] The positively charged proteins may render the negatively charged nanoparticles unstable and destabilize the conjugate via electrostatic interaction. [ 149 ] Besides, the antibody covalently functionalized nanoparticles may exhibit enhanced in vivo stability in comparison with antibody‐adsorbed nanoparticles, since the covalent linkages can better prevent the competitive displacement of antibodies by blood components. [ 266 ] However, pH‐sensitive chemical linkages (e.g., disulfide) utilized to develop responsive nanoparticles may be unstable in the blood circulation because of the complex interactions of antibody‐conjugated nanoparticles with blood components.…”

Super-resolution imaging of antibody-conjugated biodegradable periodic mesoporous organosilica nanoparticles for targeted chemotherapy of prostate cancer

“…35 Therefore, the evaluation of the surface functionality of nanoparticles is one of the most important steps for the development of potent nanomedicine with future potential in clinical applications. 36 Nevertheless, the accurate quantification of such biomolecules conjugated to each nanoparticle's surface (i.e., nanoparticle multivalency) by conventional assays using low-sensitivity instruments has been challenging to date, [37][38][39][40] and becomes more complicated when the molar concentration of the nanoparticles cannot be easily determined with high accuracy and precision. 41,42 Recently, our research group has been investigating the surface functionality to better understand the distribution/orientation/number of targeting ligands present at a single-particle level using the single-molecule dSTORM imaging technique.…”