Ekaterina S. Lisitsyna

Owing to doxorubicin’s high DNA binding affinity, doxorubicin-loaded DNA origami nanostructures (DOX-DONs) are promising nanocarriers against cancer. However, understanding the interactions between doxorubicin (DOX) and DNA origami nanostructures (DONs) is important to ensure the quality of DOX-DONs. This interaction is often taken for granted and the influence of DOX loading conditions is poorly characterized. Exploiting the inherent fluorescence of DOX, steady-state and time-resolved fluorescence anisotropy spectroscopy techniques are used for characterizing nondestructively the binding between DOX and DONs, and the purity of the formed complexes. The difference in fluorescence anisotropy between free DOX and DOX-DONs confirms the DOX-DON complex formation. Further, at loading ratios of DOX to DNA base pairs >0.5, homo-Förster resonance energy transfer (homo-FRET) between closely packed DOX molecules is observed. Moreover, time-resolved anisotropy reveals DOX aggregation on DONs at high loading ratios >1. For loading ratios >0.1, spin-filtration to remove excess free DOX is efficient and necessary, though at loading ratios >1 some DOX aggregates remain attached to the DONs. In summary, fluorescence anisotropy analysis provides more detailed information and insight into DOX-DONs compared to the regularly used fluorescence intensity-based characterization methods, and these results can help designing more efficient and safer DNA intercalator-based nanocarriers.

Owing to doxorubicin’s high DNA binding affinity, doxorubicin-loaded DNA origami nanostructures (DOX-DONs) are promising nanocarriers against cancer. However, understanding the interactions between doxorubicin (DOX) and DNA origami nanostructures (DONs) is important to ensure the quality of DOX-DONs. This interaction is often taken for granted and the influence of DOX loading conditions is poorly characterized. Exploiting the inherent fluorescence of DOX, steady-state and time-resolved fluorescence anisotropy spectroscopy techniques are used for characterizing non-destructively the binding between DOX and DONs, and the purity of formed complexes. The difference in fluorescence anisotropy between free DOX and DOX-DONs confirms the DOX-DON complex formation. Further, at loading ratios of DOX to DNA base pairs > 0.5, homo-Förster resonance energy transfer (homo-FRET) between closely packed DOX molecules is observed. Moreover, time-resolved anisotropy reveals DOX aggregation on DONs at high loading ratios > 1. For loading ratios > 0.1, spin-filtration to remove excess free DOX is efficient and necessary, though at loading ratios > 1 some DOX aggregates remain attached to the DONs. In summary, fluorescence anisotropy analysis provides more detailed information and insight into DOX-DONs compared to regularly used fluorescence intensity-based characterization methods, and these results can help designing more efficient and safer DNA intercalator-based nanocarriers.

The perovskite-inspired Cu₂AgBiI₆ (CABI) absorber shows promise for low-toxicity indoor photovoltaics. However, the carrier self-trapping in this material limits its photovoltaic performance. Herein, we examine the self-trapping mechanism in CABI by analyzing the excited-state dynamics of its absorption band at 425 nm, which is responsible for the self-trapped exciton emission, using a combination of photoluminescence and ultrafast transient absorption spectroscopies. Photoexcitation in CABI rapidly generates charge carriers in the silver iodide lattice sites, which localize into the self-trapped states and luminesce. Furthermore, a Cu-Ag-I-rich phase that exhibits similar spectral responses as CABI is synthesized, and a comprehensive structural and photophysical study of this phase provides insights into the nature of the excited states of CABI. Overall, this work explains the origin of self-trapping in CABI. This understanding will play a crucial role in optimizing its optoelectronic properties. It also encourages compositional engineering as the key to suppressing self-trapping in CABI.

For long-term live-cell fluorescence imaging and biosensing, it is crucial to work with a dye that has high fluorescence quantum yield and photostability without being detrimental to the cells. In this paper, we demonstrate that neutral boron-dipyrromethene (BODIPY)-based molecular rotors have great properties for high-light-dosage demanding live-cell fluorescence imaging applications that require repetitive illuminations. In molecular rotors, an intramolecular rotation (IMR) allows an alternative route for the decay of the singlet excited state (S₁) via the formation of an intramolecular charge transfer state (CT). The occurrence of IMR reduces the probability of the formation of a triplet state (T₁) which could further react with molecular oxygen (³O₂) to form cytotoxic reactive oxygen species, e.g., singlet oxygen (¹O₂). We demonstrate that the oxygen-related nature of the phototoxicity for BODIPY derivatives can be significantly reduced if a neutral molecular rotor is used as a probe. The studied neutral molecular rotor probe shows remarkably lower phototoxicity when compared with both the non-rotating BODIPY derivative and the cationic BODIPY-based molecular rotor in different light dosages and dye concentrations. It is also evident that the charge and localization of the fluorescent probe are as significant as the IMR in terms of the phototoxicity in a long-term live-cell imaging. Graphical abstract

Correction for ‘Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles’ by Kaisa Rautaniemi et al., Nanoscale Adv., 2022, DOI: 10.1039/d1na00755f.

Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter E_rp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.

Structural dynamics of the polyethylenimine–DNA and poly(l-lysine)–DNA complexes (polyplexes) was studied by steady-state and time-resolved fluorescence spectroscopy using the fluorescence resonance energy transfer (FRET) technique. During the formation of the DNA polyplexes, the negative phosphate groups (P) of DNA are bound by the positive amine groups (N) of the polymer. At N/P ratio 2, nearly all of the DNA’s P groups are bound by the polymer N groups: these complexes form the core of the polyplexes. The excess polymer, added to this system to increase the N/P ratio to the values giving efficient gene delivery, forms a positively charged shell around the core polyplex. We investigated whether the exchange between the core and shell regions of PEI and PLL polyplexes takes place. Our results demonstrated a clear difference between the two studied polymers. Shell PEI can replace PEIs previously attached to DNA in the polyplex core, while PLL cannot. Such a dynamic structure of PEI polyplexes compared to a more static one found for PLL polyplexes partially explains the observed difference in the DNA transfection efficiency of these polyplexes. Moreover, the time-resolved fluorescence spectroscopy revealed additional details on the structure of PLL polyplexes: in between the core and shell, there is an intermediate layer where both core and shell PLLs or their parts overlap.