Maslov, Mikhail Aleksandrovich

Shishlyannikov S.M., Zubkov I.N., Vysochinskaya V.V., Gavrilova N.V., Dobrovolskaya O.A., Elpaeva E.A., Maslov M.A., Vasin A.

Background/Objectives: The development of polymer–lipid hybrid nanoparticles (PLNs) is a promising area of research, as it can help increase the stability of cationic lipid carriers. Hybrid PLNs are core–shell nanoparticle structures that combine the advantages of both polymer nanoparticles and liposomes, especially in terms of their physical stability and biocompatibility. Natural polymers such as polyhydroxyalkanoate (PHA) can be used as a matrix for the PLNs’ preparation. Methods: In this study, we first obtained stable cationic hybrid PLNs using a cationic liposome (CL) composed of a polycationic lipid 2X3 (1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and the hydrophobic polymer mcl-PHA, which was produced by the soil bacterium Pseudomonas helmantisensis P1. Results: The new polymer-lipid carriers effectively encapsulated and delivered model mRNA-eGFP (enhanced green fluorescent protein mRNA) to BHK-21 cells. We then evaluated the role of mcl-PHA in increasing the stability of cationic PLNs in ionic solutions using dynamic light scattering data, electrophoretic mobility, and transmission electron microscopy techniques. Conclusions: The results showed that increasing the concentration of PBS (phosphate buffered saline) led to a decrease in the stability of the CLs. At high concentrations of PBS, the CLs aggregate. In contrast, the presence of isotonic PBS did not result in the aggregation of PLNs, and the particles remained stable for 120 h when stored at +4 °C. The obtained results show that PLNs hold promise for further in vivo studies on nucleic acid delivery.

Shmendel E.V., Markov O.V., Zenkova M.A., Maslov M.A.

INTRODUCTION. The use of cationic liposomes is a promising approach to the delivery of therapeutic nucleic acids to target cells because liposomes can protect nucleic acids from degradation by extracellular nucleases. However, to ensure selective delivery to the site of action, this approach needs modification, including liposome surface functionalisation with targeting ligands.AIM. This study aimed to compare the time courses of the accumulation of a fluorescent-labelled oligonucleotide (FITC-ODN), which simulated a nucleic acid-based medicinal product, in cells with the use of folate receptor-targeted (F) and conventional (L) cationic liposomes.MATERIALS AND METHODS. F- and L-liposomes were prepared using the polycationic amphiphile 2X3, the zwitterionic helper lipid DOPE, and the folate lipoconjugate F12. Physicochemical characterisation of the liposomes was performed using dynamic light scattering and transmission electron microscopy. Liposome–FITC-ODN complexes were formed at various nitrogen to phosphate (N/P) charge ratios. Flow cytometry, fluorescence microscopy, and confocal microscopy methods were used to study the accumulation of liposome–FITC-ODN complexes in human cervical carcinoma (KB-3-1) and human embryonic kidney (HEK 293) cells.RESULTS. The prepared F- and L-liposomes were spherical particles with a diameter of 75–100 nm. The authors selected the optimal N/P ratio of 2/1 to obtain complexes of F- and L-liposomes with the FITC-ODN. This N/P ratio yielded homogeneous liposome–FITC-ODN complexes having a polydispersity index below 0.200 and a size of 112.4–125.1 nm. F-liposomes were 25% more efficient than L-liposomes in FITC-ODN delivery to KB-3-1 cells at 90, 120, and 240 minutes after transfection. In the first few minutes of cell transfection, fluorescence and confocal microscopy data on the distribution of liposome–FITC-ODN complexes showed that cationic liposome fluorescence signals colocalised with FITC-ODN signals. Later, FITC-ODN accumulation in the cytoplasm was observed.CONCLUSIONS. Cationic liposomes demonstrated efficient FITC-ODN delivery into the cytoplasm of cancer cells. F-liposomes enhanced the percentage of transfected cells and improved FITC-ODN delivery compared with L-liposomes. The results obtained can be used in the further development of targeted medicinal products based on therapeutic nucleic acids and liposomes.

Vysochinskaya V., Zabrodskaya Y., Dovbysh O., Emelyanov A., Klimenko V., Knyazev N., Terterov I., Egorova M., Bogdanov A., Maslov M., Vasin A., Dubina M.

Gene silencing through RNA interference (RNAi) is a promising therapeutic approach for a wide range of disorders, including cancer. Non-viral gene therapy, using specific siRNAs against BCR-ABL1, can be a supportive or alternative measure to traditional chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKIs) therapies, given the prevalence of clinical TKI resistance. The main challenge for such approaches remains the development of the effective delivery system for siRNA tailored to the specific disease model. The purpose of this study was to examine and compare the efficiency of endosomolytic cell penetrating peptide (CPP) EB1 and PEG2000-decorated cationic liposomes composed of polycationic lipid 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2Х3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for anti-bcr-abl siRNA delivery into the K562 human CML cell line. We show that both EB1 and 2Х3-DOPE-DSPE-PEG2000 (0.62 % mol.) liposomes effectively deliver siRNA into K562 cells by endocytic mechanisms, and the use of liposomes leads to more effective inhibition of expression of the targeted gene (BCR-ABL1) and cancer cell proliferation. Taken together, these findings suggest that PEG-decorated cationic liposomes mediated siRNA delivery allows an effective antisense suppression of certain oncogenes, and represents a promising new class of therapies for CML.

Fedorovskiy A.G., Antropov D.N., Dome A.S., Puchkov P.A., Makarova D.M., Konopleva M.V., Matveeva A.M., Panova E.A., Shmendel E.V., Maslov M.A., Dmitriev S.E., Stepanov G.A., Markov O.V.

Over the past decade, mRNA-based therapy has displayed significant promise in a wide range of clinical applications. The most striking example of the leap in the development of mRNA technologies was the mass vaccination against COVID-19 during the pandemic. The emergence of large-scale technology and positive experience of mRNA immunization sparked the development of antiviral and anti-cancer mRNA vaccines as well as therapeutic mRNA agents for genetic and other diseases. To facilitate mRNA delivery, lipid nanoparticles (LNPs) have been successfully employed. However, the diverse use of mRNA therapeutic approaches requires the development of adaptable LNP delivery systems that can control the kinetics of mRNA uptake and expression in target cells. Here, we report effective mRNA delivery into cultured mammalian cells (HEK293T, HeLa, DC2.4) and living mouse muscle tissues by liposomes containing either 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) or the newly applied 1,30-bis(cholest-5-en-3β-yloxycarbonylamino)-9,13,18,22-tetraaza-3,6,25,28-tetraoxatriacontane tetrahydrochloride (2X7) cationic lipids. Using end-point and real-time monitoring of Fluc mRNA expression, we showed that these LNPs exhibited an unusually delayed (of over 10 h in the case of the 2X7-based system) but had highly efficient and prolonged reporter activity in cells. Accordingly, both LNP formulations decorated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) provided efficient luciferase production in mice, peaking on day 3 after intramuscular injection. Notably, the bioluminescence was observed only at the site of injection in caudal thigh muscles, thereby demonstrating local expression of the model gene of interest. The developed mRNA delivery systems hold promise for prophylactic applications, where sustained synthesis of defensive proteins is required, and open doors to new possibilities in mRNA-based therapies.

Bishani A., Makarova D.M., Shmendel E.V., Maslov M.A., Sen‘kova A.V., Savin I.A., Gladkikh D.V., Zenkova M.A., Chernolovskaya E.L.

In this study, the impact of different delivery systems on the cytokine-inducing, antiproliferative, and antitumor activities of short immunostimulatory double-stranded RNA (isRNA) was investigated. The delivery systems, consisting of the polycationic amphiphile 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20 tetraazahexacosan tetrahydrochloride (2X3), and the lipid-helper dioleoylphosphatidylethanolamine (DOPE), were equipped with polyethylene glycol lipoconjugates differing in molecular weight and structure. The main findings of this work are as follows: (i) significant activation of MCP-1 and INF-α, β, and γ production in CBA mice occurs under the action of isRNA complexes with liposomes containing lipoconjugates with long PEG chains, while activation of MCP-1 and INF-γ, but not INF-α or β, was observed under the action of isRNA lipoplexes containing lipoconjugates with short PEG chains; (ii) a pronounced antiproliferative effect on B16 melanoma cells in vitro, as well as an antitumor and hepatoprotective effect in vivo, was induced by isRNA pre-complexes with non-pegylated liposomes, while complexes containing lipoconjugates with long-chain liposomes were inactive; (iii) the antitumor activity of isRNA correlated with the efficiency of its accumulation in the cells and did not explicitly depend on the activation of cytokine and interferon production. Thus, the structure of the delivery system plays a vital role in determining the response to isRNA and allows for the choice of a delivery system depending on the desired effect.

Shmendel E.V., Puchkov P.A., Maslov M.A.

The delivery of therapeutic nucleic acids is a prospective method for the treatment of both inherited and acquired diseases including cancer. To achieve maximal delivery efficiency and selectivity, nucleic acids should be targeted to the cells of interest. In the case of cancer, such targeting may be provided through folate receptors overexpressed in many tumor cells. For this purpose, folic acid and its lipoconjugates are used. Compared to other targeting ligands, folic acid provides low immunogenicity, rapid tumor penetration, high affinity to a wide range of tumors, chemical stability, and easy production. Different delivery systems can utilize targeting by folate ligand including liposomal forms of anticancer drugs, viruses, and lipid and polymer nanoparticles. This review focuses on the liposomal gene delivery systems that provide targeted nucleic acid transport into tumor cells due to folate lipoconjugates. Moreover, important development step, such as rational design of lipoconjugates, folic acid content, size, and ζ-potential of lipoplexes are discussed.

Petukhov I.A., Puchkov P.A., Morozova N.G., Zenkova M.A., Maslov M.A.

Vysochinskaya V., Shishlyannikov S., Zabrodskaya Y., Shmendel E., Klotchenko S., Dobrovolskaya O., Gavrilova N., Makarova D., Plotnikova M., Elpaeva E., Gorshkov A., Moshkoff D., Maslov M., Vasin A.

The design of cationic liposomes for efficient mRNA delivery can significantly improve mRNA-based therapies. Lipoplexes based on polycationic lipid 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were formulated in different molar ratios (1:1, 1:2, 1:3) to efficiently deliver model mRNAs to BHK-21 and A549. The objective of this study was to examine the effect of 2X3-DOPE composition as well as lipid-to-mRNA ratio (amino-to-phosphate group ratio, N/P) on mRNA transfection. We found that lipoplex-mediated transfection efficiency depends on both liposome composition and the N/P ratio. Lipoplexes with an N/P ratio of 10/1 showed nanometric hydrodynamic size, positive ζ potential, maximum loading, and transfection efficiency. Liposomes 2X3-DOPE (1:3) provided the superior delivery of both mRNA coding firefly luciferase and mRNA-eGFP into BHK-21 cells and A549 cells, compared with commercial Lipofectamine MessengerMax.

Perevoshchikova K.A., Eshtukova-Shcheglova E.A., Markov O.V., Markov A.V., Chernikov I.V., Maslov M.A., Zenkova M.A.

Unsymmetric lipophilic polyamine derivatives are considered as potential antitumor agents. Here, a series of novel symmetric lipophilic polyamines (LPAs) based on norspermine and triethylenetetramine (TETA) backbones bearing alkyl substituents with different lengths (from decyl to octadecyl) at C(1) atom of glycerol were synthesized. Performed screening of the cytotoxicity of novel compounds on the panel of tumor cell lines (MCF-7, KB-3-1, B16) and non-malignant fibroblasts hFF3 in vitro revealed a correlation between the length of the aliphatic moieties in LPAs and their toxic effects - LPAs with the shortest decyl substituent were found to exhibit the highest cytotoxicity. Furthermore, norspermine-based LPAs displayed somewhat more pronounced cytotoxicity compared with their TETA-based counterparts. Further mechanistic studies demonstrated that hit LPAs containing the norspermine backbone and tetradecyl or decyl substituents efficiently induced apoptosis in KB-3-1 cells. Moreover, decyl-bearing LPA inhibited motility and enhanced adhesiveness of murine B16 melanoma cells in vitro, showing promising antimetastatic potential. Thus, developed novel symmetric norspermine-based LPAs can be considered as promising anticancer chemotherapeutic candidates.

Markov O.V., Sen’kova A.V., Mohamed I.S., Shmendel E.V., Maslov M.A., Oshchepkova A.L., Brenner E.V., Mironova N.L., Zenkova M.A.

Cell-free antitumor vaccines represent a promising approach to immunotherapy of cancer. Here, we compare the antitumor potential of cell-free vaccines based on microvesicles derived from dendritic cells (DCs) with DC- and cationic-liposome-based vaccines using a murine model of drug-resistant lymphosarcoma RLS40 in vivo. The vaccines were the following: microvesicle vaccines—cytochalasin B-induced membrane vesicles (CIMVs) obtained from DCs loaded with total tumor RNA using cholesterol/spermine-containing cationic liposomes L or mannosylated liposomes ML; DC vaccines—murine DCs loaded with total tumor-derived RNA using the same liposomes; and liposomal vaccines—lipoplexes of total tumor-derived RNA with liposomes L or ML. Being non-hepatotoxic, CIMV- and DC-based vaccines administered subcutaneously exhibited comparable potential to stimulate highly efficient antitumor CTLs in vivo, whereas liposomal vaccines were 25% weaker CTL inducers. Nevertheless, the antitumor efficiencies of the different types of the vaccines were similar: sizes of tumor nodes and the number of liver metastases were significantly decreased, regardless of the vaccine type. Notably, the booster vaccination did not improve the overall antitumor efficacy of the vaccines under the study. CIMV- and DC- based vaccines more efficiently than liposome-based ones decreased mitotic activity of tumor cells and induced their apoptosis, stimulated accumulation of neutrophil inflammatory infiltration in tumor tissue, and had a more pronounced immunomodulatory activity toward the spleen and thymus. Administration of CIMV-, DC-, and liposome-based vaccines resulted in activation of Th1/Th17 cells as well as the induction of positive immune checkpoint 4-1BBL and downregulation of suppressive immune checkpoints in a raw PD-1 >>> TIGIT > CTLA4 > TIM3. We demonstrated that cell-free CIMV-based vaccines exhibited superior antitumor and antimetastatic activity in a tumor model in vivo. The obtained results can be considered as the basis for developing novel strategies for oncoimmunotherapy.

Eshtukova-Shcheglova E.A., Perevoshchikova K.A., Eshtukov-Shcheglov A.V., Cheshkov D.A., Maslov M.A.

Objectives. Alkylated derivatives of polyamines are able to block the growth of cancer cells due to their embedding into the polyamine biosynthesis mechanisms. The study aimed to synthesize lipophilic derivatives of norspermine or triethylenetetramine based on the formation of a C–N bond during the opening of the oxirane ring by primary amines to expand a number of synthetic polyamine derivatives with antitumor activity.Methods. The starting compounds—glycidol alcoholate or epichlorohydrin—were reacted with hexadecyl bromide or sodium hexadecanolate to give glycidyl hexadecyl ether. The key reaction for the preparation of lipophilic polyamines was the amination of lipophilic epoxides with polyamines in the presence of calcium triflate. Acylation of the hydroxyl group formed during the opening of oxirane was carried out by the action of 4-dimethylaminopyridine and acetic anhydride. The introduction of an alkyl substituent in the presence of sodium hydride led to intramolecular cyclization with the formation of an oxoazolidine cycle. The regioselectivity of the oxirane ring opening reaction at the C(1) position of glycerol was confirmed by two-dimensional heteronuclear {1H,13C} nuclear magnetic resonance spectroscopy.Results. An approach to the synthesis of novel lipophilic polyamines based on the catalytic amination of epoxides was developed and tested. Compounds based on norspermine and triethylentetramine containing a hydroxyl group at the C(2) atom of the glycerin backbone were obtained. For norspermine derivatives, the hydroxyl group was modified: an acetyl substituent was introduced and a derivative containing an oxoazolidine cycle was obtained.Conclusions. The obtained lipophilic polyamines can be considered as potential antitumor agents, for which cytotoxicity against various cancer cells will be evaluated in the future.

Nichugovskiy A., Maksimova V., Trapeznikova E., Eshtukova-Shcheglova E., Ivanov I., Yakubovskaya M., Kirsanov K., Cheshkov D., Tron G.C., Maslov M.

Natural polyamines (PAs) are involved in the processes of proliferation and differentiation of cancer cells. Lipophilic synthetic polyamines (LPAs) induce the cell death of various cancer cell lines. In the current paper, we have demonstrated a new method for synthesis of LPAs via the multicomponent Ugi reaction and subsequent reduction of amide groups by PhSiH3. The anticancer activity of the obtained compounds was evaluated in the A-549, MCF7, and HCT116 cancer cell lines. For the first time, it was shown that the anticancer activity of LPAs with piperazine fragments is comparable with that of aliphatic LPAs. The presence of a diglyceride fragment in the structure of LPAs appears to be a key factor for the manifestation of high anticancer activity. The findings of the study strongly support further research in the field of LPAs and their derivatives.

Gaponova S., Patutina O., Sen’kova A., Burakova E., Savin I., Markov A., Shmendel E., Maslov M., Stetsenko D., Vlassov V., Zenkova M.

Rational combinations of sequence-specific inhibitors of pro-oncogenic miRNAs can efficiently interfere with specific tumor survival pathways, offering great promise for targeted therapy of oncological diseases. Herein, we uncovered the potential of multicomponent therapy by double or triple combinations of highly potent mesyl phosphoramidate (µ) antisense oligodeoxynucleotides targeted to three proven pro-oncogenic microRNAs—miR-17, miR-21, and miR-155. A strong synergism in the inhibition of proliferation and migration of B16 melanoma cells was demonstrated in vitro for pairs of µ-oligonucleotides, which resulted in vivo in profound inhibition (up to 85%) of lung metastases development after intravenous injection of µ-oligonucleotide-transfected B16 cells in mice. A clear benefit of µ-21-ON/µ-17-ON and µ-17-ON/µ-155-ON/µ-21-ON combination antitumor therapy was shown for the lymphosarcoma RLS40 solid tumor model. In vivo administration of the µ-17-ON/µ-155-ON/µ-21-ON cocktail into RLS40-bearing mice elicited fourfold delay of tumor growth as a result of strong inhibition of tumor mitotic activity. It was discovered that the cocktail of µ-21-ON/µ-17-ON/µ-155-ON led to a twofold decrease in total destructive changes in murine liver, which indicates both the reduction in toxic tumor burden and the absence of specific toxicity of the proposed therapy.

Nikolaeva M.E., Nechaev A.V., Shmendel E.V., Akasov R.A., Maslov M.A., Mironov A.F.

Upconverting nanoparticles have unique spectral and photophysical properties that make them suitable for development of theranostics for imaging and treating large and deep-seated tumors. Nanoparticles based on NaYF4 crystals doped with lanthanides Yb3+ and Er3+ were obtained by the high-temperature decomposition of trifluoroacetates in oleic acid and 1-octadecene. Such particles have pronounced hydrophobic properties. Therefore, to obtain stable dispersions in aqueous media for the study of their properties in vivo and in vitro, the polyethylene glycol (PEG)-glycerolipids of various structures were obtained. To increase the circulation time of PEG-lipid coated nanoparticles in the bloodstream, long-chain substituents are needed to be attached to the glycerol backbone using ether bonds. To prevent nanoparticle aggregation, an L-cysteine-derived negatively charged carboxy group should be included in the lipid molecule.

Shahbaaz M., Maslov D.A., Vatlin A.A., Danilenko V.N., Grishina M., Christoffels A.

In the current era of a pandemic, infections of COVID-19 and Tuberculosis (TB) enhance the detrimental effects of both diseases in suffering individuals. The resistance mechanisms evolving in Mycobacterium tuberculosis are limiting the efficiency of current therapeutic measures and pressurizing the stressed medical infrastructures. The bacterial efflux pumps enable the development of resistance against recently approved drugs such as bedaquiline and clofazimine. Consequently, the MmpS5-MmpL5 protein system was selected because of its role in efflux pumping of anti-TB drugs. The MmpS5-MmpL5 systems of Mycobacterium smegmatis were modelled and the virtual screening was performed using an ASINEX library of 5968 anti-bacterial compounds. The inhibitors with the highest binding affinities and QSAR based highest predicted inhibitory concentration were selected. The MmpS5-MmpL5 associated systems with BDE_26593610 and BDD_27860195 showed highest inhibitory parameters. These were subjected to 100 ns Molecular Dynamics simulations and provided the validation regarding the interaction studies. The in vitro studies demonstrated that the BDE_26593610 and BDD_27860195 can be considered as active inhibitors for M. smegmatis MmpS5-MmpL5. The outcomes of this study can be utilized in other experimentation aimed at drug design and discovery against the drug resistance strains of M. tuberculosis.

Ihnatsyeu-Kachan A., Sharko O., Bekish A., Saichuk A., Zhogla V., Abashkin V., Ulashchik E., Shcharbin D., Le Goff W., Kontush A., Guillas I., Shmanai V., Kim S.

Wen J., Xu J., Hong M., Li W., Li T.

Auchynnikava T., Äärelä A., Moisio O., Liljenbäck H., Andriana P., Iqbal I., Laine T., Palani S., Lehtimäki J., Rajander J., Salo H., Airaksinen A.J., Virta P., Roivainen A.

Simeonova K.B., Koleva A.I., Petkova-Yankova N.I., Zlatanova A.R., Lozanova V., Nikolova R.D., Petkov P.S.

The current study is a continuation of our previous investigations into the radical homodimeric reaction mechanism of 3-acetylcoumarin. In the current study, the effects of different substituents on the coumarin ring of 3-acetylcoumarin are investigated both experimentally and theoretically. Several 3-acetylcoumarin derivatives (substituted at C-6, C-7, and C-8) were tested in the optimized reaction conditions under ultrasound irradiation, and biscoumarin species were isolated and characterized. The elucidation of the substituent’s effect was further investigated by means of DFT calculations (free-energy calculations, NBO analysis), both in the initial substituted coumarins and in the formed radicals. It was observed that the presence of substituents at the C-6 and C-8 positions in the coumarin moiety would not affect significantly the formation of a radical, while a group at position C-7 could either stabilize or destabilize the formed radical depending on the electronic properties of the substituent.

Sen’kova A.V., Bishani A., Savin I.A., Zenkova M.A., Chernolovskaya E.L.

Previously, we described a 19-base pair double-stranded RNA with 3'-trinucleotide overhangs, acting as immunostimulatory RNA (isRNA). This molecule demonstrated notable antiproliferative effects on cancer cells, inhibited tumor growth, and elicited immunostimulatory and antiviral responses by inducing cytokine and interferon production. Within this study, we compared the efficiency of lung fibrosis development, initiated in mice by BLM or LPS using different schemes of induction. Then we compared the effect of isRNA used in a preventive or therapeutic regimen on the development of fibrosis in selected BLM- and LPS-induced mouse models and showed that isRNA can be used in pathological conditions accompanied by the development of inflammation and the risk of fibrosis formation, without adverse side effects. Prophylactic regimen of isRNA application is beneficial for prevention of the development of pulmonary fibrosis.

Kanp T., Dhuri A., M B., Rode K., Aalhate M., Paul P., Nair R., Singh P.K.

Liu S., Tan B., Wang F., Yu Y.

Glioblastoma (GBM) remains one of the most aggressive and treatment-resistant brain tumors, necessitating innovative therapeutic approaches. Polymer-based nanotechnology has emerged as a promising solution, offering precise drug delivery, enhanced blood-brain barrier (BBB) penetration, and adaptability to the tumor microenvironment (TME). This review explores the diverse applications of polymeric nanoparticles (NPs) in GBM treatment, including delivery of chemotherapeutics, targeted therapeutics, immunotherapeutics, and other agents for radiosensitization and photodynamic therapy. Recent advances in targeted delivery and multifunctional polymer highlight their potential to overcome the challenges that GBM brought, such as heterogeneity of the tumor, BBB limitation, immunosuppressive TME, and consideration of biocompatibility and safety. Meanwhile, the future directions to address these challenges are also proposed. By addressing these obstacles, polymer-based nanotechnology represents a transformative strategy for improving GBM treatment outcomes, paving the way for more effective and patient-specific therapies.

Soghrati S., Varshosaz J., Rostami M., Mirian M.

Combinational therapy to defend triple negative breast cancer (TNBC) by affecting different cellular pathways concomitantly, has gained attention recently. In the present study, co-delivery of dasatinib and miR30a by means...

Yadav K., Ebenezer Gnanakani S.P., Kumar Sahu K., Sucheta, Dubey A., Minz S., Raza W., Pradhan M.

Askarizadeh A., Vahdat-Lasemi F., Karav S., Kesharwani P., Sahebkar A.

Klabenkova K., Zakhryamina A., Burakova E., Bizyaev S., Fokina A., Stetsenko D.

Nowadays, nucleic acid derivatives capable of modulating gene expression at the RNA level have gained widespread recognition as promising therapeutic agents. A suitable degree of biological stability of oligonucleotide therapeutics is required for in vivo application; this can be most expeditiously achieved by the chemical modification of the internucleotidic phosphate group, which may also affect their cellular uptake, tissue distribution and pharmacokinetics. Our group has previously developed a strategy for the chemical modification of the phosphate group via the Staudinger reaction on a solid phase of the intermediate dinucleoside phosphite triester and a range of, preferably, electron deficient organic azides such as sulfonyl azides during automated solid-phase DNA synthesis according to the conventional β-cyanoethyl phosphoramidite scheme. Polyfluoro compounds are characterized by unique properties that have prompted their extensive application both in industry and in scientific research. We report herein the synthesis and isolation of novel oligodeoxyribonucleotides incorporating internucleotidic perfluoro-1-octanesulfonyl phosphoramidate or 2,2,2-trifluoroethanesulfonyl phosphoramidate groups. In addition, novel oligonucleotide derivatives with fluorinated zwitterionic phosphate mimics were synthesized by a tandem methodology, which involved (a) the introduction of a carboxylic ester group at the internucleotidic position via the Staudinger reaction with methyl 2,2-difluoro-3-azidosulfonylacetate; and (b) treatment with an aliphatic diamine, e.g., 1,1-dimethylethylenediamine or 1,3-diaminopropane. It was further shown that the polyfluoro oligonucleotides obtained were able to form complementary duplexes with either DNA or RNA, which were not significantly differing in stability from the natural counterparts. Long-chain perfluoroalkyl oligonucleotides were taken up into cultured human cells in the absence of a transfection agent. It may be concluded that the polyfluoro oligonucleotides described here can represent a useful platform for designing oligonucleotide therapeutics.

Shmendel E.V., Buyanova A.O., Markov O.V., Morozova N.G., Zenkova M.A., Maslov M.A.

New cationic amphiphiles containing lactose or D-mannose residues were synthesized and cationic liposomes with 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) were obtained. The cytotoxicity and transfection activity of new carbohydrate-containing amphiphiles and cationic liposomes against HEK 293, BHK and BHK IR-780 cells were studied. It has been shown that cationic amphiphiles effectively deliver only short fluorescein-labeled oligodeoxyribonucleotide into eukaryotic cells, while cationic liposomes formed by lactose containing amphiphile and DOPE effectively mediate the transport of short oligonucleotide and small interfering RNA and were non-toxic to cells. The resulting cationic amphiphiles can be used for intracellular delivering of nucleic acids both individually and part of cationic liposomes.

Dement’eva O.V., Shishmakova E.M., Ivchenko A.V., Staltsov M.S., Markova A.A., Rudoy V.M.

It has been shown that associates of cationic glycerolipid (CGL), rac-N-{4-[(2-ethoxy-3-octadecyloxyprop-1-yl)oxycarbonyl]butyl}-N'-methylimidazolium iodide, which has a pronounced antitumor effect, can be used for the solubilization of two hydrophobic biologically active compounds (curcumin and capsaicin) and as templates in the sol–gel synthesis of silica mesoporous container particles (MCPs). The thermodynamic characteristics of solubilization are determined, and it is shown that this process contributes to a significant increase in the solubility of both hydrophobic drugs in water. The hydrolytic condensation of tetraethoxysilane in the presence of CGL associates containing curcumin or capsaicin leads to the formation of MCPs characterized by a narrow size distribution and a high content of encapsulated drugs. This combination of the stages of the synthesis and loading of MCPs is of undoubted interest in relation to the nanoencapsulation of cationic glycerolipids (including in combination with other drugs).

Shmendel E.V., Buyanova A.O., Markov O.V., Morozova N.G., Zenkova M.A., Maslov M.A.

Objective: The development of systems for targeted delivery of nucleic acids (NAs) is necessary to ensure their selective transport to the site of therapeutic action. The aim of this work was to synthesize carbohydrate-modified amphiphiles containing a spermine residue, required for compaction and binding to NAs, as well as a diglyceride residue for forming lipid aggregates and a carbohydrate residue (lactose or D-mannose) for improving the hydrophilic–lipophilic balance of the molecule. The lactose residue can serve as a targeting ligand for NA delivery into liver hepatocytes, and the D-mannose residue can perform specific NA transport into dendritic cells and macrophages. Methods: New carbohydrate-modified cationic amphiphiles were obtained by organic synthesis, and their aqueous dispersions or cationic liposomes were prepared. Cytotoxicity of the cationic amphiphiles and liposomes was performed using the MTT assay on HEK 293 and BHK cell lines in the absence of fetal bovine serum (FBS). Complexes of the cationic amphiphiles or liposomes with NAs (FITC-ODN, pDNA, and siRNA) were formed at various component ratios (N/P), and the efficiency of transfection in HEK 293 and BHK IR-780 cells was assessed by flow cytometry. Results and Discussion: New cationic amphiphiles containing lactose or D-mannose residues were synthesized. The cationic amphiphiles, whatever the structure of their carbohydrate residue, effectively deliver a short FITC-ODN into HEK293 cells in the presence of FBS, and are nontoxic. The cationic liposome formed by the lactose-containing amphiphile and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) successfully delivers short NAs (FITC-ODN and siRNA) both in the absence and in the presence of serum in the culture media. Conclusions: The obtained carbohydrate-modified cationic amphiphiles, both individually and as component of cationic liposomes, hold promise to be used as systems for the delivery of short nucleic acids in further development of drugs for gene therapy.

Zabrodskaya Y.A., Gavrilova N.V., Elpaeva E.A., Lozhkov A.A., Vysochinskaya V.V., Dobrovolskaya O.A., Dovbysh O.V., Zimmerman E.L., Dav P.N., Brodskaia A.V., Sakhenberg E.I., Shaldzhyan A.A., Demaev A.A., Maslov M.A., Vasin A.V.

Yadava S.K., Reddy B.P., Prausnitz M.R., Cicerone M.T.

Opsomer L., Jana S., Mertens I., Cui X., Hoogenboom R., Sanders N.

SaRNA was formulated with six different polymer carriers to generate saRNA-polyplexes. After screening for transfection efficiency, compared to a lipid-based standard, the best performing ones were characterized in detail and administered to mice.

Schober G.B., Story S., Arya D.P.

AbstractWith the recent success of lipid nanoparticle (LNP) based SARS-CoV-2 mRNA vaccines, the potential for RNA therapeutics has gained widespread attention. LNPs are promising non-viral delivery vectors to protect and deliver delicate RNA therapeutics, which are ineffective and susceptible to degradation alone. While food and drug administration (FDA) approved formulations have shown significant promise, benchmark lipid formulations still require optimization and improvement. In addition, the translatability of these formulations for several different RNA cargo sizes has not been compared under the same conditions. Herein we analyze “gold standard” lipid formulations for encapsulation efficiency of various non-specific RNA cargo lengths representing antisense oligonucleotides (ASO), small interfering RNA (siRNA), RNA aptamers, and messenger RNA (mRNA), with lengths of 10 bases, 21 base pairs, 96 bases, 996 bases, and 1929 bases, respectively. We evaluate encapsulation efficiency as the percentage of input RNA encapsulated in the final LNP product (EEinput%), which shows discrepancy with the traditional calculation of encapsulation efficiency (EE%). EEinput% is shown to be < 50% for all formulations tested, when EE% is consistently > 85%. We also compared formulations for LNP size (Z-average) and polydispersity index (PDI). LNP size does not appear to be strongly influenced by cargo size, which is a counterintuitive finding. Thoughtful characterization of LNPs, in parallel with consideration of in vitro or in vivo behavior, will guide design and optimization for better understanding and improvement of future RNA therapeutics.

Zhang H., Vandesompele J., Braeckmans K., De Smedt S.C., Remaut K.

This review discusses DNases and RNases present in the human body, strategies to reduce the degradation of gene therapeutics after administration and available methods to follow nuclease activity in buffer and living cells.

Huang P., Deng H., Wang C., Zhou Y., Chen X.

AbstractMessenger RNA (mRNA)‐based therapy has emerged as a powerful, safe, and rapidly scalable therapeutic approach that involves technologies for both mRNA itself and the delivery vehicle. Although there are some unique challenges for different applications of mRNA therapy, a common challenge for all mRNA therapeutics is the transport of mRNA into the target cell cytoplasm for sufficient protein expression. In this review, we focus on the behaviors of nanotechnology‐mediated mRNA delivery systems at the cellular level, which has not been comprehensively reviewed yet. First, we introduce the four main therapeutic applications of mRNA, including immunotherapy, protein replacement therapy, genome editing, and cellular reprogramming. Second, we summarize common types of mRNA cargo and mRNA delivery systems. Third, we highlight strategies to enhance mRNA delivery efficiency during the cellular trafficking process, including accumulation to the cell, internalization into the cell, endosomal escape, release of mRNA from the nanocarrier, and translation of mRNA into protein. Finally, we present the challenges and opportunities for the development of nanotechnology‐mediated mRNA delivery system. We hope this review can provide new insights into the future fabrication of mRNA nanocarriers with desirable cellular trafficking performance.This article is protected by copyright. All rights reserved

Philipp J., Dabkowska A., Reiser A., Frank K., Krzysztoń R., Brummer C., Nickel B., Blanchet C.E., Sudarsan A., Ibrahim M., Johansson S., Skantze P., Skantze U., Östman S., Johansson M., et. al.

Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), cationic ionizable lipid (CIL), cholesterol (chol), and mRNA. Yet the mechanism of pH-dependent response that is believed to cause endosomal release of LNPs is not well understood. Here, we show that eGFP (enhanced green fluorescent protein) protein expression in the mouse liver mediated by the ionizable lipids DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD) ranks MC3 ≥ KC2 > DD despite similar delivery of mRNA per cell in all cell fractions isolated. We hypothesize that the three CIL-LNPs react differently to pH changes and hence study the structure of CIL/chol bulk phases in water. Using synchrotron X-ray scattering a sequence of ordered CIL/chol mesophases with lowering pH values are observed. These phases show isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal H II and bicontinuous cubic Pn3m symmetry. If polyadenylic acid, as mRNA surrogate, is added to CIL/chol, excess lipid coexists with a condensed nucleic acid lipid H II c phase. The next-neighbor distance in the excess phase shows a discontinuity at the Fd3m inverse micellar to inverse hexagonal H II transition occurring at pH 6 with distinctly larger spacing and hydration for DD vs. MC3 and KC2. In mRNA LNPs, DD showed larger internal spacing, as well as retarded onset and reduced level of DD-LNP-mediated eGFP expression in vitro compared to MC3 and KC2. Our data suggest that the pH-driven Fd3m- H II transition in bulk phases is a hallmark of CIL-specific differences in mRNA LNP efficacy.

Fedorovskiy A.G., Burakov A.V., Terenin I.M., Bykov D.A., Lashkevich K.A., Popenko V.I., Makarova N.E., Sorokin I.I., Sukhinina A.P., Prassolov V.S., Ivanov P.V., Dmitriev S.E.

Abstract In response to stress stimuli, eukaryotic cells typically suppress protein synthesis. This leads to the release of mRNAs from polysomes, their condensation with RNA-binding proteins, and the formation of non-membrane-bound cytoplasmic compartments called stress granules (SGs). SGs contain 40S but generally lack 60S ribosomal subunits. It is known that cycloheximide, emetine, and anisomycin, the ribosome inhibitors that block the progression of 80S ribosomes along mRNA and stabilize polysomes, prevent SG assembly. Conversely, puromycin, which induces premature termination, releases mRNA from polysomes and stimulates the formation of SGs. The same effect is caused by some translation initiation inhibitors, which lead to polysome disassembly and the accumulation of mRNAs in the form of stalled 48S preinitiation complexes. Based on these and other data, it is believed that the trigger for SG formation is the presence of mRNA with extended ribosome-free segments, which tend to form condensates in the cell. In this study, we evaluated the ability of various small-molecule translation inhibitors to block or stimulate the assembly of SGs under conditions of severe oxidative stress induced by sodium arsenite. Contrary to expectations, we found that ribosome-targeting elongation inhibitors of a specific type, which arrest solitary 80S ribosomes at the beginning of the mRNA coding regions but do not interfere with all subsequent ribosomes in completing translation and leaving the transcripts (such as harringtonine, lactimidomycin, or T-2 toxin), completely prevent the formation of arsenite-induced SGs. These observations suggest that the presence of even a single 80S ribosome on mRNA is sufficient to prevent its recruitment into SGs, and the presence of extended ribosome-free regions of mRNA is not sufficient for SG formation. We propose that mRNA entry into SGs may be mediated by specific contacts between RNA-binding proteins and those regions on 40S subunits that remain inaccessible when ribosomes are associated.

Zhang W., Pfeifle A., Lansdell C., Frahm G., Cecillon J., Tamming L., Gravel C., Gao J., Thulasi Raman S.N., Wang L., Sauve S., Rosu-Myles M., Li X., Johnston M.J.

In recent years, lipid nanoparticles (LNPs) have emerged as a revolutionary technology for vaccine delivery. LNPs serve as an integral component of mRNA vaccines by protecting and transporting the mRNA payload into host cells. Despite their prominence in mRNA vaccines, there remains a notable gap in our understanding of the potential application of LNPs for the delivery of DNA vaccines. In this study, we sought to investigate the suitability of leading LNP formulations for the delivery of plasmid DNA (pDNA). In addition, we aimed to explore key differences in the properties of popular LNP formulations when delivering either mRNA or DNA. To address these questions, we compared three leading LNP formulations encapsulating mRNA- or pDNA-encoding firefly luciferase based on potency, expression kinetics, biodistribution, and immunogenicity. Following intramuscular injection in mice, we determined that RNA-LNPs formulated with either SM-102 or ALC-0315 lipids were the most potent (all p-values < 0.01) and immunogenic (all p-values < 0.05), while DNA-LNPs formulated with SM-102 or ALC-0315 demonstrated the longest duration of signal. Additionally, all LNP formulations were found to induce expression in the liver that was proportional to the signal at the injection site (SM102: r = 0.8787, p < 0.0001; ALC0315: r = 0.9012, p < 0.0001; KC2: r = 0.9343, p < 0.0001). Overall, this study provides important insights into the differences between leading LNP formulations and their applicability to DNA- and RNA-based vaccinations.

Shchaslyvyi A.Y., Antonenko S.V., Tesliuk M.G., Telegeev G.D.

In the realm of gene therapy, a pivotal moment arrived with Paul Berg’s groundbreaking identification of the first recombinant DNA in 1972. This achievement set the stage for future breakthroughs. Conditions once considered undefeatable, like melanoma, pancreatic cancer, and a host of other ailments, are now being addressed at their root cause—the genetic level. Presently, the gene therapy landscape stands adorned with 22 approved in vivo and ex vivo products, including IMLYGIC, LUXTURNA, Zolgensma, Spinraza, Patisiran, and many more. In this comprehensive exploration, we delve into a rich assortment of 16 drugs, from siRNA, miRNA, and CRISPR/Cas9 to DNA aptamers and TRAIL/APO2L, as well as 46 carriers, from AAV, AdV, LNPs, and exosomes to naked mRNA, sonoporation, and magnetofection. The article also discusses the advantages and disadvantages of each product and vector type, as well as the current challenges faced in the practical use of gene therapy and its future potential.

Zhang W., Jiang Y., He Y., Boucetta H., Wu J., Chen Z., He W.

Messenger RNA (mRNA) is the template for protein biosynthesis and is emerging as an essential active molecule to combat various diseases, including viral infection and cancer. Especially, mRNA-based vaccines, as a new type of vaccine, have played a leading role in fighting against the current global pandemic of COVID-19. However, the inherent drawbacks, including large size, negative charge, and instability, hinder its use as a therapeutic agent. Lipid carriers are distinguishable and promising vehicles for mRNA delivery, owning the capacity to encapsulate and deliver negatively charged drugs to the targeted tissues and release cargoes at the desired time. Here, we first summarized the structure and properties of different lipid carriers, such as liposomes, liposome-like nanoparticles, solid lipid nanoparticles, lipid-polymer hybrid nanoparticles, nanoemulsions, exosomes and lipoprotein particles, and their applications in delivering mRNA. Then, the development of lipid-based formulations as vaccine delivery systems was discussed and highlighted. Recent advancements in the mRNA vaccine of COVID-19 were emphasized. Finally, we described our future vision and perspectives in this field.

Zubkov I.N., Vysochinskaya V.V., Kashina A.V., Shishlyannikov S.M.

Nanoparticles based on biodegradable polymers find numerous applications in medicine as substances for intracellular drug delivery. Biosynthetically produced poly-3-hydroxyalkanoates (P3HAs) are among the most promising polymers of a lipid nature. In particular, polyhydroxybutyrate and polyhydroxyvalerate (scl-poly-3-hydroxyalkanoates) are widely used compounds, which are soluble only in organochlorine solvents. The use of organochlorine solvents faces several obstacles, since such chemicals may exhibit carcinogenic effects on the human body. However, the P3Has compounds consisting of hydroxybutyric acid residues with 6–14 carbon atoms in the main chain (mcl-poly-3-hydroxyalkanoates) are soluble not only in CHCl3 or CH2Cl2 but also in paraffins. These hydrocarbons, such as n-hexane, can be easily separated from aqueous solutions and are not known to exhibit high cytotoxicity. Consequently, the application of mcl-poly-3-hydroxyalkanoates can prevent the contamination of prepared dosage forms with organochlorine compounds. To this end, a methodology for the synthesis of mcl-P3HA nanoparticles stabilized with the Tween 80 nonionic surfactant was proposed. The ratio between the concentration of P3HA and the detergent was optimized. The present study revealed that the obtained particles have an average size of 200±90 nm and a zeta potential of -17±5 mV. Upon investigating the stability of the particle solution at 4 °C, it was found that the nanoparticles did not alter their size and zeta potential for 90 days. The fluorescence microscopy method showed that they could be delivered into BHK-21 cells within 2 h. In addition, the nanoparticles synthesized had no toxic effect on BHK-21 cells at a concentration of up to 200 μg/mL.

Szewczyk-Roszczenko O.K., Roszczenko P., Shmakova A., Finiuk N., Holota S., Lesyk R., Bielawska A., Vassetzky Y., Bielawski K.

Endocytosis is one of the major ways cells communicate with their environment. This process is frequently hijacked by pathogens. Endocytosis also participates in the oncogenic transformation. Here, we review the approaches to inhibit endocytosis, discuss chemical inhibitors of this process, and discuss potential clinical applications of the endocytosis inhibitors.

Bishani A., Makarova D.M., Shmendel E.V., Maslov M.A., Sen‘kova A.V., Savin I.A., Gladkikh D.V., Zenkova M.A., Chernolovskaya E.L.

In this study, the impact of different delivery systems on the cytokine-inducing, antiproliferative, and antitumor activities of short immunostimulatory double-stranded RNA (isRNA) was investigated. The delivery systems, consisting of the polycationic amphiphile 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20 tetraazahexacosan tetrahydrochloride (2X3), and the lipid-helper dioleoylphosphatidylethanolamine (DOPE), were equipped with polyethylene glycol lipoconjugates differing in molecular weight and structure. The main findings of this work are as follows: (i) significant activation of MCP-1 and INF-α, β, and γ production in CBA mice occurs under the action of isRNA complexes with liposomes containing lipoconjugates with long PEG chains, while activation of MCP-1 and INF-γ, but not INF-α or β, was observed under the action of isRNA lipoplexes containing lipoconjugates with short PEG chains; (ii) a pronounced antiproliferative effect on B16 melanoma cells in vitro, as well as an antitumor and hepatoprotective effect in vivo, was induced by isRNA pre-complexes with non-pegylated liposomes, while complexes containing lipoconjugates with long-chain liposomes were inactive; (iii) the antitumor activity of isRNA correlated with the efficiency of its accumulation in the cells and did not explicitly depend on the activation of cytokine and interferon production. Thus, the structure of the delivery system plays a vital role in determining the response to isRNA and allows for the choice of a delivery system depending on the desired effect.

Grisolia A., Dell’Olio G., Spadafora A., De Santo M., Morelli C., Leggio A., Pasqua L.

Due to the ever-growing global population, it is necessary to develop highly effective processes that minimize the impact of human activities and consumption on the environment. The levels of organic and inorganic contaminants have rapidly increased in recent years, posing a threat to ecosystems. Removing these toxic pollutants from the environment is a challenging task that requires physical, chemical, and biological methods. An effective solution involves the use of novel engineered materials, such as silica-based nanostructured materials, which exhibit a high removal capacity for various pollutants. The starting materials are also thermally and mechanically stable, allowing for easy design and development at the nanoscale through versatile functionalization procedures, enabling their effective use in pollutant capture. However, improvements concerning mechanical properties or applicability for repeated cycles may be required to refine their structural features. This review focuses on hybrid/composite polymer-silica nanostructured materials. The state of the art in nanomaterial synthesis, different techniques of functionalization, and polymer grafting are described. Furthermore, it explores the application of polymer-modified nanostructured materials for the capture of heavy metals, dyes, hydrocarbons and petroleum derivatives, drugs, and other organic compounds. The paper concludes by offering recommendations for future research aimed at advancing the application of polymer-silica nanostructured materials in the efficiency of pollutant uptake.

Zheng L., Bandara S.R., Tan Z., Leal C.

RNA therapeutics have the potential to resolve a myriad of genetic diseases. Lipid nanoparticles (LNPs) are among the most successful RNA delivery systems. Expanding their use for the treatment of more genetic diseases hinges on our ability to continuously evolve the design of LNPs with high potency, cellular-specific targeting, and low side effects. Overcoming the difficulty of releasing cargo from endocytosed LNPs remains a significant hurdle. Here, we investigate the fundamental properties of nonviral RNA nanoparticles pertaining to the activation of topological transformations of endosomal membranes and RNA translocation into the cytosol. We show that, beyond composition, LNP fusogenicity can be prescribed by designing LNP nanostructures that lower the energetic cost of fusion and fusion–pore formation with a target membrane. The inclusion of structurally active lipids leads to enhanced LNP endosomal fusion, fast evasion of endosomal entrapment, and efficacious RNA delivery. For example, conserving the lipid make-up, RNA–LNPs having cuboplex nanostructures are significantly more efficacious at endosomal escape than traditional lipoplex constructs.