Advances in molecular biology have revolutionized the use of messenger RNA (mRNA) as a therapeutic. The concept of nucleic acid therapy with mRNA originated in 1990 when Wolff et al. reported successful expression of proteins in target organs by direct injection of either plasmid DNA or mRNA. It took decades to bring the transfection efficiency of mRNA closer to that of DNA. The next few decades were dedicated to turning in vitro-transcribed (IVT) mRNA from a promising delivery tool for gene therapy into a full-blown therapeutic modality, which changed the biotech market rapidly. Hundreds of clinical trials are currently underway using mRNA for prophylaxis and therapy of infectious diseases and cancers, in regenerative medicine, and genome editing. The potential of IVT mRNA to induce an innate immune response favors its use for vaccination and immunotherapy. Nonetheless, in non-immunotherapy applications, the intrinsic immunostimulatory activity of mRNA directly hinders the desired therapeutic effect since it can seriously impair the target protein expression. Targeting the same innate immune factors can increase the effectiveness of mRNA therapeutics for some indications and decrease it for others, and vice versa. The review aims to present the innate immunity-related ‘barriers’ or ‘springboards’ that may affect the development of immunotherapies and non-immunotherapy applications of mRNA medicines.

MRNA-based therapeutics have been found to be a promising treatment strategy in immunotherapy, gene therapy, and cancer treatments. Effectiveness of mRNA therapeutics depends on the level and duration of a desired protein’s expression, which is determined by various cis- and trans-regulatory elements of the mRNA. Sequences of 5′ and 3′ untranslated regions (UTRs) are responsible for translational efficiency and stability of mRNA. An optimal combination of the regulatory sequences allows researchers to significantly increase the target protein’s expression. Using both literature data and previously obtained experimental data, we chose six sequences of 5′UTRs (adenoviral tripartite leader [TPL], HBB, rabbit β-globin [Rabb], H4C2, Moderna, and Neo2) and five sequences of 3′UTRs (mtRNR-EMCV, mtRNR-AES, mtRNR-mtRNR, BioNTech, and Moderna). By combining them, we constructed 30 in vitro transcribed RNAs encoding firefly luciferase with various combinations of 5′- and 3′UTRs, and the resultant bioluminescence was assessed in the DC2.4 cell line at 4, 8, 24, and 72 h after transfection. The cellular data enabled us to identify the best seven combinations of 5′- and 3′UTRs, whose translational efficiency was then assessed in BALB/c mice. Two combinations of 5′- and 3′UTRs (5′Rabb-3′mtRNR-EMCV and 5′TPL-3′Biontech) led to the most pronounced increase in the luciferase amount in the in vivo experiment in mice. Subsequent analysis of the stability of the mRNA indicated that the increase in luciferase expression is explained primarily by the efficiency of translation, not by the number of RNA molecules. Altogether, these findings suggest that 5′UTR-and-3′UTR combinations 5′Rabb-3′mtRNR- EMCV and 5′TPL-3′Biontech lead to high expression of target proteins and may be considered for use in preventive and therapeutic modalities based on mRNA.

A novel protocol for topical influenza immunization is reported. CaCO₃ carriers are used to deliver vaccine into hair follicles aiming to target perifollicular antigen presenting cells. This approach represents a promising alternative to injections.

The design of cargo carriers with high biocompatibility, unique morphological characteristics, and capability of strong bonding of fluorescent dye is highly important for the development of a platform for smart imaging and diagnostics. In this paper, BODIPY-doped silica nanoparticles were prepared through a “one-pot” soft-template method using a sol-gel process. Several sol-gel precursors have been used in sol-gel synthesis in the presence of soft-template to obtain the silica-based materials with the most appropriate morphological features for the immobilization of BODIPY molecules. Obtained silica particles have been shown to be non-cytotoxic and can be effectively internalized into the cervical cancer cell line (HeLa). The described method of synthesis allows us to obtain silica-based carriers with an immobilized fluorescent dye that provide the possibility for real-time imaging and detection of these carriers.

The behavior and migration of human mesenchymal stromal cells (hMSCs) are focal points of research in the biomedical field. One of the major aspects is potential therapy using hMCS, but at present, the safety of their use is still controversial owing to limited data on changes that occur with hMSCs in the long term. Fluorescent photoconvertible proteins are intensively used today as “gold standard” to mark the individual cells and study single-cell interactions, migration processes, and the formation of pure lines. A crucial disadvantage of this method is the need for genetic modification of the primary culture, which casts doubt on the possibility of exploring the resulting clones in personalized medicine. Here we present a new approach for labeling and tracking hMSCs without genetic modification based on the application of cell-internalizable photoconvertible polyelectrolyte microcapsules (size: 2.6 ± 0.5 μm). These capsules were loaded with rhodamine B, and after thermal treatment, exhibited fluorescent photoconversion properties. Photoconvertible capsules demonstrated low cytotoxicity, did not affect the immunophenotype of the hMSCs, and maintained a high level of fluorescent signal for at least seven days. The developed approach was tested for cell tracking for four days and made it possible to trace the destiny of daughter cells without the need for additional labeling.

Reliable cell labeling and tracking techniques are imperative for elucidating the intricate and ambiguous interactions between mesenchymal stromal cells (MSCs) and tumors. Here, we explore fluorescent photoconvertible nanoengineered vesicles to study mMSC migration in brain tumors. These 3 μm sized vesicles made of carbon nanoparticles, Rhodamine B (RhB), and polyelectrolytes are readily internalized by cells. The dye undergoes photoconversion under 561 nm laser exposure with a fluorescence blue shift upon demand. The optimal laser irradiation duration for photoconversion was 0.4 ms, which provided a maximal blue shift of the fluorescent signal label without excessive laser exposure on cells. Vesicles modified with an extra polymer layer demonstrated enhanced intracellular uptake without remarkable effects on cell viability, motility, or proliferation. The optimal ratio of 20 vesicles per mMSC was determined. Moreover, the migration of individual mMSCs within 2D and 3D glioblastoma cell (EPNT-5) colonies over 2 days and in vivo tumor settings over 7 days were traced. Our study provides a robust nanocomposite platform for investigating MSC–tumor dynamics and offers insights into envisaged therapeutic strategies. Photoconvertible vesicles also present an indispensable tool for studying complex fundamental processes of cell–cell interactions for a wide range of problems in biomedicine.