Protein affinity reagents (e.g., antibodies) are often used for basic research, diagnostics, separations, and disease therapy. Although a lot of "synthetic" protein affinity reagents have been developed as a cost-effective alternative to antibodies, their low biocompatibility is a considerable problem for clinical application. Lipid nanoparticles (LNP) represent a highly biocompatible drug delivery agent. However, little has been reported that LNP itself works as a protein affinity reagent in living animals. Here, LNP is engineered for binding to and neutralizing a target toxic peptide in living animals by multifunctionalization with amino acid derivatives. Multifunctionalized LNP (MF-LNP) is prepared using amino acid derivative-conjugated lipids. Optimized MF-LNP exhibits nanomolar affinity to the target toxic peptide and inhibits toxic peptide-dependent hemolysis and cytotoxicity. In addition, MF-LNP captures and neutralizes the toxic peptide after intravenous injection in the bloodstream; in addition, MF-LNP does not release the toxic peptide in the accumulated organ. These results reveal the potential of using LNP as a highly biocompatible protein affinity reagent such as an antidote.
Multifunctional synthetic polymers can bind to target molecules and are therefore widely investigated in diagnostics, drug delivery carriers, and separation carriers. Because these polymers are synthesized from nonbiological components, purification processes (e.g., chromatography, dialysis, extraction, and centrifugation) must be conducted after the synthesis. Although several purification methods are used for polymer purification, few reports have revealed the influence of purification process on the functions of polymer. In this study, we demonstrated that the characteristics, function, and stability of synthetic polymer depend on the purification process. N-Isopropylacrylamide-based polymer nanoparticles (NPs) and melittin (i.e., honey bee venom) were used as a model of synthetic polymer and target toxic peptide, respectively. Synthesized NPs were purified by dialysis in methanol, acetone precipitation, or centrifugation. NPs purified by dialysis in ultrapure water were used as control NPs. Then, NP size, surface charge, toxin neutralization effect, and stability were determined. NP size did not considerably change by purification with centrifugation; however, it decreased by purification using dialysis in methanol and acetone precipitation compared with that of control NPs. The ζ-potential of NPs changed after each purification process compared with that of control NPs. The melittin neutralization efficiency of NPs depended on the purification process; i.e., it decreased by acetone precipitation and increased by dialysis in methanol and centrifugation compared with that of control NPs. Of note, the addition of methanol and acetone decreased NP stability. These studies implied the importance of considering the effect of the purification method on synthetic polymer function.
We developed abiotic polymer ligand (PL)-decorated lipid nanoparticles (LNPs) to improve PL mobility, decrease aggregation after capturing the target, and increase the blood circulation time to achieve highly effective toxin neutralization in vivo.
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