Magnetic Nanoworm-Based Screening of Yeast Library for Anti-PEG Nanobodies.

IF 4.5 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL
Shashikant Ray, Sarah Jacques, Utibeabasi Ettah, Shaodong Dai, Hanmant K Gaikwad, Robert I Scheinman, Krishna M G Mallela, Dmitri Simberg
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引用次数: 0

Abstract

PEGylation is commonly utilized to modify nanoparticles, vaccines, therapeutic proteins, and biomaterials. The increased use of PEG-containing cosmetics and medicines results in anti-PEG immunoglobulins in humans, including IgG, IgM, and IgE types. Recent studies have shown that high-affinity antibodies against PEG can be isolated through immunization. These antibodies have been used in immunoassays and for decorating PEGylated nanoparticles with targeting ligands. However, conventional antibodies have limitations, such as their large size and potential stability issues. Here, we developed a protocol to isolate anti-PEG nanobodies (Nbs) from a yeast library. We prepared ∼60 nm fluorescently labeled PEGylated and non-PEGylated iron oxide nanoworms for negative and positive magnetic selection, followed by multiparameter flow cytometry sorting. The representative Nb clone exhibited highly specific binding to PEGylated liposomes and nanoparticles and was able to compete with commercial antibackbone PEG IgG but not with anti-methoxy PEG IgG for PEG binding. The Nb showed a micromolar dissociation constant, no aggregation, high thermal stability, and reversible structural stability. This work represents a proof-of-concept screening and isolation of anti-PEG Nbs.

基于磁性纳米虫的抗peg纳米体酵母文库筛选
聚乙二醇化通常用于修饰纳米颗粒、疫苗、治疗蛋白和生物材料。含有聚乙二醇的化妆品和药物的使用增加,导致人类产生抗聚乙二醇免疫球蛋白,包括IgG、IgM和IgE类型。最近的研究表明,可以通过免疫分离出高亲和力的PEG抗体。这些抗体已用于免疫测定和修饰聚乙二醇化纳米粒子与靶向配体。然而,传统的抗体有局限性,比如它们的大尺寸和潜在的稳定性问题。在这里,我们开发了一种从酵母文库中分离抗peg纳米体(Nbs)的方案。我们制备了约60 nm荧光标记的聚乙二醇化和非聚乙二醇化氧化铁纳米虫,用于阴性和阳性磁选择,然后进行多参数流式细胞术分选。具有代表性的Nb克隆显示出与聚乙二醇化脂质体和纳米颗粒的高度特异性结合,并且能够与商业抗骨架PEG IgG竞争,但不能与抗甲氧基PEG IgG竞争PEG结合。Nb具有微摩尔解离常数、不聚集、高热稳定性和可逆结构稳定性。这项工作代表了抗peg Nbs的概念验证筛选和分离。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Molecular Pharmaceutics
Molecular Pharmaceutics 医学-药学
CiteScore
8.00
自引率
6.10%
发文量
391
审稿时长
2 months
期刊介绍: Molecular Pharmaceutics publishes the results of original research that contributes significantly to the molecular mechanistic understanding of drug delivery and drug delivery systems. The journal encourages contributions describing research at the interface of drug discovery and drug development. Scientific areas within the scope of the journal include physical and pharmaceutical chemistry, biochemistry and biophysics, molecular and cellular biology, and polymer and materials science as they relate to drug and drug delivery system efficacy. Mechanistic Drug Delivery and Drug Targeting research on modulating activity and efficacy of a drug or drug product is within the scope of Molecular Pharmaceutics. Theoretical and experimental peer-reviewed research articles, communications, reviews, and perspectives are welcomed.
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