Influence of Nuclear Localization Sequences on the Intracellular Fate of Gold Nanoparticles

Abstract

Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLSfunctionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.

Surface enhanced Raman scattering for probing cellular biochemistry

“… 82 Combined SERS and electron microscopy data indicated that the nanoparticles are processed differently in the endolysosomal system when they carry an NLS, even when they do not undergo endosomal escape. 82 In the situation where endosomal escape does not take place, the endosomal molecular composition and interaction of the modified gold nanoparticles resembles that of unmodified nanoparticles. At the same time, the SERS spectra indicated special molecular properties of the intracellular agglomerates, which was related to their ‘attempt’ to engage in endosomal escape.…”

“…76,80 The success in probing the intracellular environment in this case depends greatly on whether and which molecules are used for potential functionalization, how they change the uptake dynamics and how accessible the particle surface remains. 58,81,82 Cells incubated with gold nanoparticles functionalized with polyadenine show different interactions within the same incubation period as non-functionalized particles. 81 While the bare gold nanoparticles interact mainly with lipids after 4 h, and for longer incubation times lipid bands decrease and protein and 15 nucleic acid related bands increase, functionalization with polyadenine results in a decreased interaction with lipids at shorter times and faster appearance of protein related bands in the spectra, suggesting that this functionalization facilitates cellular uptake of nanoparticles and induces a faster formation of endosomes.…”

“…Experiments that can reveal the interaction of SERS nanoprobes with the cells at the level of the cellular ultrastructure, particularly electron microscopy and nanotomography based on X-ray microscopy have proven indispensable for understanding and controlling the targeting of organelles. 7,82 SERS spectra from the nucleus can probe changes that occur when cells undergo specific treatments or programmed cell death. The effect of hyperoxia in mouse lung fibroblasts cells, 85 and cell damage upon UV irradiation 84 were followed by SERS in the nucleus, using gold nanocubes with NLS.…”

“…For nanoparticles that made it into the nucleus, bands related to nucleic acid vibrations, such as thymine at 753 cm −1 or cytosine and adenine at 1246 cm −1 appeared more often. 82 These combined SERS and electron microscopy experiments show that optimum incubation conditions for nuclear localization must be identified in order to utilize SERS signals from the nucleus. Experiments that can reveal the interaction of SERS nanoprobes with the cells at the level of the cellular ultrastructure, particularly electron microscopy and nanotomography based on X-ray microscopy have proven indispensable for understanding and controlling the targeting of organelles.…”

Nucleus-Targeting Phototherapy Nanodrugs for High-Effective Anti-Cancer Treatment

“…Phototherapeutic nanodrugs can be passively targeted to the nucleus by changing surface properties and size because of the highly physicochemical flexibility of nanomaterials ( Jiang et al, 2020 ; Xu et al, 2020 ). More importantly, active targeting of the nucleus can also be achieved by modifying nucleus-targeting groups such as cell-penetrating peptides [e.g., transactivator of transcription (TAT)] ( Su et al, 2020 ; Tietz et al, 2022 ), the specific nuclear localization signal/sequence (NLS) peptide ( Zelmer et al, 2020 ; Drescher et al, 2021 ), and DNA aptamers ( Dong et al, 2018 ; Yang et al, 2018 ; Zeng et al, 2020 ) on phototherapeutic nanodrugs. Apart from this, ROS generated by phototherapeutic nanodrugs can also damage the membrane structure to increase permeability ( Chen et al, 2021 ; Su et al, 2021 ; Chen et al, 2022 ).…”