Search

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. [ABSTRACT FROM AUTHOR]

The spindle checkpoint arrests cells in metaphase until all chromosomes are properly attached to the chromosome segregation machinery. Thereafter, the anaphase promoting complex (APC/C) is activated and chromosome segregation can take place. Cells remain arrested in mitosis for hours in response to checkpoint activation, but not indefinitely. Eventually, they adapt to the checkpoint and proceed along the cell cycle. In yeast, adaptation requires the phosphorylation of APC/C. Here, we show that the protein phosphatase PP2ACdc55 dephosphorylates APC/C, thereby counteracting the activity of the mitotic kinase Cdc28. We also observe that the key regulator of Cdc28, the mitotic cyclin Clb2, increases before cells adapt and is then abruptly degraded at adaptation. Adaptation is highly asynchronous and takes place over a range of several hours. Our data suggest the presence of a double negative loop between PP2ACdc55 and APC/CCdc20 (i.e., a positive feedback loop) that controls APC/CCdc20 activity. The circuit could guarantee sustained APC/CCdc20 activity after Clb2 starts to be degraded. [ABSTRACT FROM AUTHOR]

Bothtrans- andcis-[PtCl2(NH3)(L)] compounds have been synthesized, L representing either the imino ether HN=C(OMe)Me having aZorEconfiguration at the C=N double bond, or the cyclic ligandsand(compounds1-4fortransgeometry and5-8forcisgeometry, respectively). The cyclic ligands mimic the imino ether ligands but, differently from imino ethers, cannot undergo change of configuration. In a panel of human tumor cells,transcompounds inhibit growth much more than transplatin. Moreover, compound1in most cases is less active than2, and1and2are less active than3and4, respectively. Forciscompounds with imino ethers, the activity is reduced (5) or unaffected (6) with respect to cisplatin. Moreover, unliketranscompounds, substitution of cyclic ligands (7,8) for imino ethers (5,6) generally decreases the activity. This determines, for compounds with cyclic ligands, an unusual inversion of thecisgeometry requirement for activity of platinum(II) species. Importantly,1-4and5-8partially circumvent the multifocal cisplatin resistance of A2780cisR cells, and1-4also overcome resistance from reduced uptake of 41McisR cells. DNA interaction regioselectivity of1-4and5-8is not substantially modified with respect to transplatin and cisplatin. However, both imino ethers and cyclic ligands slow down the DNA interstrand cross-link reaction, (E)-HN=C(OMe)Me anddecreasing also its extent. Therefore, DNA interaction of1-4and5-8appears to be characterized by persistent monoadducts (1-4), and by monoadducts and/or intrastrand cross-links structurally different from those of cisplatin (5-8). This study demonstrates that ligand configuration modulates the activity of bothtransandciscompounds, and supports the development of platinum drugs based on their coordination chemistry to combat cisplatin resistance. [ABSTRACT FROM AUTHOR]