Adenoviruses represent exceptional candidates for wide-ranging therapeutic applications, from vectors for gene therapy to oncolytics for cancer treatments. The first ever commercial gene therapy medicine was based on a recombinant adenovirus vector, while most recently, adenoviral vectors have proven critical as vaccine platforms in effectively controlling the global coronavirus pandemic. Here, we discuss factors involved in adenovirus cell binding, entry, and trafficking; how they influence efficiency of adenovirus-based vectors; and how they can be manipulated to enhance efficacy of genetically modified adenoviral variants. We focus particularly on endocytosis and how different adenovirus serotypes employ different endocytic pathways to gain cell entry, and thus, have different intracellular trafficking pathways that subsequently trigger different host antiviral responses. In the context of gene therapy, the final goal of the adenovirus vector is to efficiently deliver therapeutic transgenes into the target cell nucleus, thus allowing its functional expression. Aberrant or inefficient endocytosis can impede this goal, therefore, it should be considered when designing and constructing adenovirus-based vectors.

The low seroprevalent human adenovirus type 26 (HAdV26)-based vaccine vector was the first adenovirus-based vector to receive marketing authorization from European Commission. HAdV26-based vaccine vectors induce durable humoral and cellular immune responses and, as such, represent a highly valuable tool for fighting infectious diseases. Despite well-described immunogenicity in vivo, the basic biology of HAdV26 still needs some refinement. The aim of this study was to determine the pro-inflammatory cytokine profile of epithelial cells infected with HAdV26 and then investigate the underlying molecular mechanism. The expression of studied genes and proteins was assessed by quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Confocal microscopy was used to visualize HAdV26 cell uptake. We found that HAdV26 infection in human epithelial cells triggers the expression of pro-inflammatory cytokines and chemokines, namely IL-6, IL-8, IL-1β, and TNF-α, with the most pronounced difference shown for IL-6. We investigated the underlying molecular mechanism and observed that HAdV26-induced IL-6 gene expression is αvβ3 integrin dependent and NF-κB mediated. Our findings provide new data regarding pro-inflammatory cytokine and chemokine expression in HAdV26-infected epithelial cells, as well as details concerning HAdV26-induced host signaling pathways. Information obtained within this research increases our current knowledge of HAdV26 basic biology and, as such, can contribute to further development of HAdV26-based vaccine vectors.

Newly designed and synthesized diarylethene (DAE) derivatives with aliphatic amine sidearms and one with two pyrenes, revealed excellent photo-switching property of central DAE core in MeOH and water. The only exception was bis-pyrene analogue, its DAE core very readily photochemically closed, but reversible opening completely hampered by aromatic stacking interaction of pyrene(s) with cyclic DAE. In this process, pyrene fluorescence showed to be a reliable monitoring method, an open form characterized by strong emission at 480 nm (typical for pyrene-aggregate), while closed form emitted weakly at 400 nm (typical for pyrene-DAE quenching). Only open DAE-bis-pyrene form interacted measurably with ds-DNA/RNA by flexible insertion in polynucleotide grooves, while self-stacked closed form did not bind to DNA/RNA. For the same steric reasons, flexible open DAE-bis-pyrene form was bound to at least three different binding sites at bovine serum albumin (BSA), while rigid, self-stacked closed form interacted dominantly with only one BSA site. Preliminary screening of antiproliferative activity against human lung carcinoma cell line A549 revealed that all DAE-derivatives are non-toxic. However, bis-pyrene analogue efficiently entered cells and located in the cytoplasm, whereby irradiation by light (315–400 nm) resulted in a strong, photo-induced cytotoxic effect, typical for pyrene-related singlet oxygen species production.

The 2- and 2,7- substituted para-N-methylpyridinium pyrene cations show high-affinity intercalation into ds-DNAs, whereas their non-methylated analogues interacted with ds-DNA/RNA only in the protonated form (at pH 5), but not at physiological conditions (pH 7). The fluorescence from non-methylated analogues was strongly dependent on the protonation of the pyridines; consequently, they act as fluorescence ratiometric probes for simultaneous detection of both ds-DNA and BSA at pH 5, relying on the ratio between intensities at 420 nm (BSA specific) and 520 nm (DNA specific), whereby exclusively ds-DNA sensing could be switched-off by adjustment to pH 7. Only methylated, permanently charged pyrenes show photoinduced cleavage of circular DNA, attributed to pyrene-mediated irradiation-induced production of singlet oxygen. Consequently, the moderate toxicity of these cations against human cell lines is strongly increased upon irradiation. Detailed studies revealed increased total ROS production in cells treated by the compounds studied, accompanied by cell swelling and augmentation of cellular complexity. The most photo-active 2-para-N-methylpyridinium pyrene showed significant localization at mitochondria, its photo-bioactivity likely due to mitochondrial DNA damage. Other derivatives were mostly non-selectively distributed between various cytoplasmic organelles, thus being less photoactive.

Background and purpose: Diethynylarene-linked bis(triarylborane) tetracations can be used as probes for fluorimetric and Raman sensing of biomacromolecules, as well as promising theragnostic agents. Among them, bis(triarylborane) fluorophore (TAB3), when bonded to Ag nanoparticles (NP), stood out with specific properties such as Raman signal enhancemen of the TAB3 dye in a cuvette. However, TAB3 dye - nanoparticle composites have not been studied in biological systems. For this reason, questions arose as to whether different types of metal nanoparticles (Au or Ag-based) with different coatings (negatively charged citrate or neutral PVP) could be efficiently stained with the TAB3 dye in a cuvette. The aim of this research was to examine Au and Ag nanoparticles of similar size (20-25 nm) with different stabilizers for their cellular uptake, cytotoxicity in the dark and under visible light radiation, to characterize the interactions of nanoparticles with the TAB3 fluorophore, and to study NP-TAB3 composites in cells, evaluate their intracellular staining, as well as possible photoinduced release and biological activity. Materials and methods: The binding constants of Au- and Ag- based nanoparticles with TAB3 were determined by fluorimetric titrations. The cytotoxic effect of NPs was determined by the survival of A549 cells (MTT assay). Cellular uptake of both NP and NP-TAB3 composites were performed by live cell imaging experiments. Results: The Au- or Ag-based NPs with different coatings bind to the TAB3 with high affinity. These NPs, as well as TAB3-NP complexes, efficiently enter living human cells, accumulating in cytoplasm with no apparent selectivity for a particular organelle. Even prolonged 3-day treatment with the NPs studied did not show any toxic effect on the cells. Bioimaging studies in cells revealed that the TAB3-NP complex does not intracellularly dissociate; the previously reported photo-bioactivity of TAB3 is completely inhibited by binding to NPs. Conclusion: Au- and Ag NPs were non-covalently stained by TAB3, irrespective of the different coatings, with similar binding affinities. Emission from TAB3 is strongly quenched by the NPs, but not completely. Experiments on living human cells revealed that neither free NPs, nor their composites with TAB3, were toxic. Bioimaging studies by confocal microscopy revealed that all NPs efficiently enter living cells within 90 min. Colocalization experiment with simultaneous collection of data in the reflection and fluorescence modes demonstrated that the TAB3 dye remained bound to NPs inside cells. Strong irradiation of TAB3-NP inside cells with a 457 nm laser did not yield any damage to the cells, at variance with our previously shown very strong photo-bioactivity of the TAB3 dye alone. Thus, binding of a chromophore to a nanoparticle can inhibit the chromophore’s ability to undergo photo-induced singlet oxygen production, consequently blocking its photo-bioactivity.

The innovative design of a triarylborane (TB)-dye with one NMe₂-alkylated (propargylated) group and one NMe₂ group yielded a system that is both an NMe₂ π-donor and an inductive NMe₂-alkyl cationic acceptor. Consequently, the new TB-dye was highly sensitive to a “click” reaction with an azide-substituted lysine side chain (yielding TB-lysine), resulting in a bathochromic shift of emission of 100 nm. In addition, fluorene attached to the lysine C-terminus showed FRET with the TB-chromophore, also sensitive to interactions with targets. Both the TB-dye and TB-lysine showed high affinities towards both DNA and proteins, reporting binding by an opposite fluorimetric response for DNA/RNA (quenching) vs. BSA (increase). Thus, the novel TB-dye is an ideal fluorimetric probe for orthogonal incorporation into bio-targets by “click” reactions due to fluorescence reporting of the progress of the “click” reaction and further sensing of the binding site composition. The TB-dye is moderately toxic to human cell lines after 2–3 days of exposure, but efficiently enters cells in 90 min, being non-toxic at short exposure. The most important product of the “click” reaction, TB-lysine, was non-toxic to cells and showed equal distribution between mitochondria and lysosomes. Further studies would focus particularly on the very convenient monitoring of the progress of “click” conjugation of the TB-dye with biorelevant targets inside living cells by confocal microscopy.