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Directly observing intracellular nanoparticle formation with nanocomputed tomography.

ABSTRACT: Directly observing intracellular nanostructure formation remains challenging. In this work, using a rationally designed small-molecule 4-nitrobenzyl carbamate-Cys(SEt)-Asp-Asp-Phe(iodine)-2-cyano-benzothiazole (NBC-Iod-CBT), we directly observed intracellular nanoparticle formation with nanocomputed tomography (nano-CT). In vitro, upon glutathione reduction and nitroreductase (NTR) cleavage, NBC-Iod-CBT undergoes a CBT-Cys click condensation reaction to self-assemble nanoparticles Iod-CBT-NPs with an average linear absorption coefficient (LAC) value of 0.182 ± 0.078 ?m-1 to x-ray. Nano-CT imaging of the NBC-Iod-CBT-treated, NTR-overexpressing HeLa cells showed the existence of Iod-CBT-NPs in their cytoplasm with an average LAC value of 0.172 ± 0.032 ?m-1 We anticipate that our strategy could help people to deeply understand the formation mechanism of intracellular nanostructures in the near future.

SUBMITTER: Zhang M

PROVIDER: S-EPMC7608822 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Directly observing intracellular nanoparticle formation with nanocomputed tomography.

Zhang Miaomiao M Guan Yong Y Dang Zheng Z Zhang Pinggen P Zheng Zhen Z Chen Liang L Kuang Wen W Wang Chenchen C Liang Gaolin G

Science advances 20201023 43

Directly observing intracellular nanostructure formation remains challenging. In this work, using a rationally designed small-molecule 4-nitrobenzyl carbamate-Cys(SEt)-Asp-Asp-Phe(iodine)-2-cyano-benzothiazole (NBC-Iod-CBT), we directly observed intracellular nanoparticle formation with nanocomputed tomography (nano-CT). In vitro, upon glutathione reduction and nitroreductase (NTR) cleavage, NBC-Iod-CBT undergoes a CBT-Cys click condensation reaction to self-assemble nanoparticles Iod-CBT-NPs wi ...[more]

PMID: 33097531

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Project description:Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chemically attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at determining the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is observed 16 h after cellular entry, most likely due to degradation by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core composition and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to determine that as the SNA structures are broken down over the 24 h time course of the experiment, the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degradation bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome.

Dataset Information

Directly observing intracellular nanoparticle formation with nanocomputed tomography.

Publications

Directly observing intracellular nanoparticle formation with nanocomputed tomography.

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