Ca²⁺-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca²⁺. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca²⁺-triggering “character”.

Gadolinium (Gd)-containing fullerenols are perspective agents for magnetic resonance imaging and cancer research. They combine the unique paramagnetic properties of Gd with solubility in water, low toxicity and antiradical activity of fullerenols. We compared the bioeffects of two Gd-containing fullerenols with a different number of oxygen groups—20 and 42: Gd@C₈₂O₂₀H₁₄ and Gd@C₈₂O₄₂H₃₂. The bioluminescent bacteria-based assay was applied to monitor the toxicity of fullerenols, bioluminescence was applied as a signal physiological parameter, and bacterial enzyme-based assay was used to evaluate the fullerenol effects on enzymatic intracellular processes. Chemiluminescence luminol assay was applied to monitor the content of reactive oxygen species (ROS) in bacterial and enzymatic media. It was shown that Gd@C₈₂O₄₂H₃₂ and Gd@C₈₂O₂₀H₁₄ inhibited bacterial bioluminescence at >10⁻¹ and >10⁻² gL⁻¹, respectively, revealing a lower toxicity of Gd@C₈₂O₄₂H₃₂. Low-concentration (10⁻³–10⁻¹ gL⁻¹) bacterial bioluminescence activation by Gd@C₈₂O₄₂H₃₂ was observed, while this activation was not found under exposure to Gd@C₈₂O₂₀H₁₄. Additional carboxyl groups in the structure of Gd@C₈₂O₄₂H₃₂ were determined by infrared spectroscopy and confirmed by quantum chemical calculations. The groups were supposed to endow Gd@C₈₂O₄₂H₃₂ with higher penetration ability through the cellular membrane, activation ability, lower toxicity, balancing of the ROS content in the bacterial suspensions, and lower aggregation in aqueous media.

Gd@C₈₂O_xH_y endohedral complexes for advanced biomedical applications (computer tomography, cancer treatment, etc.) were synthesized using high-frequency arc plasma discharge through a mixture of graphite and Gd₂O₃ oxide. The Gd@C₈₂ endohedral complex was isolated by high-efficiency liquid chromatography and consequently oxidized with the formation of a family of Gd endohedral fullerenols with gross formula Gd@C₈₂O₈(OH)₂₀. Fourier-transformed infrared (FTIR) spectroscopy was used to study the structure and spectroscopic properties of the complexes in combination with the DFTB3 electronic structure calculations and infrared spectra simulations. It was shown that the main IR spectral features are formed by a fullerenole C₈₂ cage that allows one to consider the force constants at the DFTB3 level of theory without consideration of gadolinium endohedral ions inside the carbon cage. Based on the comparison of experimental FTIR and theoretical DFTB3 IR spectra, it was found that oxidation of the C₈₂ cage causes the formation of Gd@C₈₂O₂₈H₂₀, with a breakdown of the integrity of the parent C₈₂ cage with the formation of pores between neighboring carbonyl and carboxyl groups. The Gd@C₈₂O₆(OOH)₂(OH)₁₈ endohedral complex with epoxy, carbonyl and carboxyl groups was considered the most reliable fullerenole structural model.

Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 μM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer–virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 10⁹ PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer–virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account.