Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht 3584, Netherlands.
Proc Natl Acad Sci U S A. 2024 Mar 12;121(11):e2307803120. doi: 10.1073/pnas.2307803120. Epub 2024 Mar 4.
Lipid nanoparticle (LNP) formulations are a proven method for the delivery of nucleic acids for gene therapy as exemplified by the worldwide rollout of LNP-based RNAi therapeutics and mRNA vaccines. However, targeting specific tissues or cells is still a major challenge. After LNP administration, LNPs interact with biological fluids (i.e., blood), components of which adsorb onto the LNP surface forming a layer of biomolecules termed the "biomolecular corona (BMC)" which affects LNP stability, biodistribution, and tissue tropism. The mechanisms by which the BMC influences tissue- and cell-specific targeting remains largely unknown, due to the technical challenges in isolating LNPs and their corona from complex biological media. In this study, we present a new technique that utilizes magnetic LNPs to isolate LNP-corona complexes from unbound proteins present in human serum. First, we developed a magnetic LNP formulation, containing >40 superparamagnetic iron oxide nanoparticles (IONPs)/LNP, the resulting LNPs containing iron oxide nanoparticles (IOLNPs) displayed a similar particle size and morphology as LNPs loaded with nucleic acids. We further demonstrated the isolation of the IOLNPs and their corresponding BMC from unbound proteins using a magnetic separation (MS) system. The BMC profile of LNP from the MS system was compared to size exclusion column chromatography and further analyzed via mass spectrometry, revealing differences in protein abundances. This new approach enabled a mild and versatile isolation of LNPs and its corona, while maintaining its structural integrity. The identification of the BMC associated with an intact LNP provides further insight into LNP interactions with biological fluids.
脂质纳米颗粒 (LNP) 制剂是一种经过验证的核酸传递方法，用于基因治疗，例如全球范围内推出的基于 LNP 的 RNAi 治疗药物和 mRNA 疫苗。然而，靶向特定组织或细胞仍然是一个主要挑战。LNP 给药后，LNP 与生物流体（即血液）相互作用，其中的成分会吸附在 LNP 表面上，形成一层称为“生物分子冠（BMC）”的生物分子，这会影响 LNP 的稳定性、生物分布和组织趋向性。由于从复杂的生物介质中分离 LNP 和它们的冠冕存在技术挑战，因此 BMC 影响组织和细胞特异性靶向的机制在很大程度上仍不清楚。在这项研究中，我们提出了一种新技术，利用磁性 LNP 从人血清中未结合的蛋白质中分离 LNP-冠冕复合物。首先，我们开发了一种包含 >40 个超顺磁性氧化铁纳米颗粒 (IONP)/LNP 的磁性 LNP 制剂，所得到的含有氧化铁纳米颗粒的 LNP（IOLNP）显示出与负载核酸的 LNP 相似的粒径和形态。我们进一步证明了使用磁性分离 (MS) 系统从未结合的蛋白质中分离 IOLNP 和它们相应的 BMC。MS 系统中 LNP 的 BMC 图谱与排阻色谱柱进行了比较，并通过质谱进一步分析，揭示了蛋白质丰度的差异。这种新方法能够温和且灵活地分离 LNP 及其冠冕，同时保持其结构完整性。与完整 LNP 相关的 BMC 的鉴定为 LNP 与生物流体的相互作用提供了进一步的深入了解。
