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Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations
Pharmaceutics ( IF 5.4 ) Pub Date : 2020-10-16 , DOI: 10.3390/pharmaceutics12100979
Ehsan Suleiman 1, 2 , Julia Mayer 2 , Elisabeth Lehner 2 , Bianca Kohlhauser 2, 3 , Alexandra Katholnig 2 , Mirjam Batzoni 2, 4 , Dominik Damm 5 , Vladimir Temchura 5 , Andreas Wagner 1 , Klaus Überla 5 , Karola Vorauer-Uhl 2
Affiliation  

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles.

中文翻译:

使用 EDC/Sulfo-NHS 化学将类天然 HIV-1 包膜三聚体与脂质体结合:要求和限制

在过去的几年中,脂质体上的类天然人类免疫缺陷病毒 1 型包膜 (HIV-1 Env) 三聚体的展示引起了广泛关注。目前,可用的方法已经能够制备出前所未有的质量的 Env-脂质体缀合物。然而,这些协议要求对 Env 三聚体进行标记和/或携带特定的功能组。出于这个原因,我们研究了 N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺/N-羟基磺基琥珀酰亚胺 (EDC/磺基-NHS) 化学物质共价结合无标签、非功能化的类天然 Env 三聚体的潜力。羧基功能化脂质体的表面。脂质体的物理完整性和免疫原构象的保存需要基于β-巯基乙醇的受控使用的微调两步法。Env 三聚体的展示严格限于带正电荷的活化脂质体,即具有正 zeta 电位的脂质体,其表面带有胺反应性 Sulfo-NHS 酯。与此一致,发现共轭高度依赖于离子强度和 pH 值。总体而言,我们已经确定了静电预浓缩(即,带负电的 Env 三聚体和通过静电吸引建立的带正电的脂质体之间的紧密接近)对于共轭反应的进行至关重要。本研究强调了潜在可扩展的基于 EDC/Sulfo-NHS 的方法的要求和局限性,并为进一步研究脂质体表面上无标签、非功能化的天然类 Env 三聚体的受控缀合奠定了坚实的基础,和其他纳米粒子。
更新日期:2020-10-17
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