Preparation of Temperature-Responsive Antibody–Nanoparticles by RAFT-Mediated Grafting from Polymerization

Abstract

Herein, we report the preparation of temperature-responsive antibody–nanoparticles by the direct polymerization of N-isopropylacrylamide (NIPAAm) from immunoglobulin G (IgG). To this end, a chain transfer agent (CTA) was introduced into IgG, followed by the precipitation polymerization of NIPAAm in an aqueous medium via reversible addition–fragmentation chain transfer polymerization above the lower critical solution temperature (LCST). Consequently, antibody–polymer particles with diameters of approximately 100–200 nm were formed. Owing to the entanglement of the grafted polymers via partial chemical crosslinking, the antibody–nanoparticles maintained their stability even at temperatures below the LCST. Further, the dispersed nanoparticles could be collected by thermal precipitation above the LCST. Additionally, the antibody–nanoparticles formulation could maintain its binding constant and exhibited a good resistance against enzymatic treatment. Thus, the proposed antibody–nanoparticles can be useful for maximizing the therapeutic potential of antibody–drug conjugates or efficacies of immunoassays and antibody recovery and recycling.

A Comparative Study of “Grafting to” and “Grafting from” Conjugation Methods for the Preparation of Antibody-Temperature-Responsive Polymer Conjugates

“…In some reported studies, a chain transfer agent (CTA) was introduced into bovine serum albumin (BSA) and lysozyme by reversible addition–fragmentation chain transfer polymerization (RAFT). − This demonstrated that the protein functionality of the conjugates was retained after the NIPAAm polymerization from proteins and the introduction of PNIPAAm by living radical polymerization. We have also succeeded in preparing antibody-temperature-responsive polymer conjugates by introducing CTA into antibodies and performing NIPAAm polymerization with antibody as the initiation point of polymerization . This conjugate that was prepared by the GF method showed nanoparticle morphology, which was also reported to be useful in improving various physical properties including stability against enzymes, high antigen–antibody reaction activity, and applicability for different medical applications, including drug delivery systems (DDSs).…”

“…On the other hand, in IgG−PNIPAAm prepared by the GF method, a wide band widening range appeared between 50 and 250 kDa, as mentioned before in our previous studies. 1,10,30,31 Similarly, the polymer introduction ratio was calculated to be 61%, which is higher than that of the GT method. Compared to the GT method, GF is less affected by steric hindrance and more efficient in polymer introduction, as polymers are directly introduced by polymerization from monomers.…”

“…IgG−PNIPAAm Conjugate Preparation by the GF Method. 30,31 Synthesis was performed according to our previously established reports, as we allowed conjugation between NHS-CTA and IgG, and an alkaline buffer was used to facilitate carbodiimide chemistry (Scheme 1). The buffer was prepared by dissolving NaHCO 3 (105 mg, 1.25 mmol) in ultrapure water (resistivity value: 18.2 Ω cm, 20 mL).…”

“…We have also succeeded in preparing antibody-temperature-responsive polymer conjugates by introducing CTA into antibodies and performing NIPAAm polymerization with antibody as the initiation point of polymerization. 31 This conjugate that was prepared by the GF method showed nanoparticle morphology, which was also reported to be useful in improving various physical properties including stability against enzymes, high antigen−antibody reaction activity, and applicability for different medical applications, including drug delivery systems (DDSs). However, there are no previous comparative studies between the GT and GF methods, which can give an indication whether antibody-polymer conjugates produced by the GF method are inferior to antibody-polymer conjugates produced by the GT method or vice versa.…”

“…Field flow fractionation measurements from our previous case study, combined with the results of this experiment, suggest that the molecular weight of the polymer in the conjugate, prepared using the GF method, is in the tens of thousands. 31 Characterization of IgG−PNIPAAm Using DLS. DLS measurements were performed to confirm the change in particle size as a result of polymer conjugation.…”