Exosomes facilitate mRNA and siRNA delivery using cationic liposomes 2X3-DOPE into rat heart mesenchymal cells in vitro



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Abstract

Aim. The delivery of nucleic acids to mesenchymal stem cells (MSCs), which are utilized as model objects in in vitro experiments or as therapeutic agents in the fields of regenerative medicine and oncology, is an actively developing area of research. Existing non-viral delivery systems either lack sufficient effectiveness or exhibit high toxicity to MSCs. Therefore, the development of new carriers has become an urgent priority.

Objective. To demonstrate the feasibility of delivering model mRNA and small interfering RNA (siRNA) to rat heart MSCs in vitro using original cationic liposomes 2X3-DOPE (1:3 mol.), and to evaluate the effect of exosomes incorporated into hybrid nanoparticles with 2X3-DOPE on the efficiency of RNA delivery.

Materials and methods. Exosomes were isolated using a standard ultracentrifugation technique, followed by characterization of the obtained vesicles through Western blotting, transmission electron microscopy, atomic force microscopy (AFM), and measurement of the hydrodynamic diameter using dynamic light scattering (DLS). siRNA was chemically synthesized, while mRNA was obtained via in vitro transcription. Complexes of liposomes or hybrid nanoparticles with RNA were prepared by mixing, and the properties of the resulting particles were assessed using DLS and AFM. To evaluate the efficiency of RNA delivery to rat heart MSCs derived from both healthy and ischemic myocardium, we employed fluorescence microscopy, laser scanning confocal microscopy, and flow cytometry.

Results. Complexes of cationic liposomes 2X3-DOPE (1:3 mol.) with mRNA, as well as 2X3-DOPE modified with DSPE-PEG2000 (0.62% mol.) complexed with siRNA, were successfully prepared and characterized. It was demonstrated that 2X3-DOPE is ineffective for mRNA delivery to rat cardiac MSCs, whereas hybrid nanoparticles incorporating exosomes based on these liposomes exhibited up to 40% transfection efficiency. Additionally, 2X3-DOPE modified with DSPE-PEG2000 (0.62% mol.) was effective for siRNA delivery to rat cardiac MSCs, achieving up to 90% transfection efficiency, while the use of hybrid nanoparticles based on this formulation resulted in 100% transfected cells, with more than a twofold increase in siRNA content within the cells, as indicated by the average fluorescence intensity.

Conclusion. Cationic liposomes 2X3-DOPE (1:3 mol.) modified with DSPE-PEG2000 (0.62% mol.) are promising vehicles for siRNA delivery to mesenchymal stem cells, both independently and in combination with exosomes. The inclusion of exosomes in hybrid nanoparticles based on 2X3-DOPE enhances the transfection efficiency of both mRNA and siRNA in rat cardiac MSCs in vitro.

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About the authors

Olesya V. Dovbysh

Almazov National Medical Research Centre; Peter the Great Saint-Petersburg Polytechnic University; Smorodintsev Research Institute of Influenza

Email: lesya.dovbysh@mail.ru
ORCID iD: 0009-0005-0924-3118
SPIN-code: 7885-7580
Russian Federation

Vera V. Vysochinskaya

Almazov National Medical Research Centre; Peter the Great Saint-Petersburg Polytechnic University; Smorodintsev Research Institute of Influenza

Email: veravv2509@gmail.com
ORCID iD: 0000-0003-3533-2606
SPIN-code: 2662-5700
Russian Federation

Nina V. Gavrilova

Peter the Great Saint-Petersburg Polytechnic University; Smorodintsev Research Institute of Influenza

Email: daughtervgater@gmail.com
ORCID iD: 0000-0002-7825-9130
SPIN-code: 1238-1989
Russian Federation

Pavel M. Docshin

Almazov National Medical Research Centre; Institute of Cytology Russian Academy of Sciences

Email: dokshin_pm@almazovcentre.ru
ORCID iD: 0000-0002-0182-009X
SPIN-code: 9896-3742
Russian Federation

Ekaterina G. Nikitina

Almazov National Medical Research Centre

Email: purrpurr@list.ru
ORCID iD: 0009-0009-0407-3307
Russian Federation

Aleksandr S. Klochev

St. Petersburg State University

Email: klochev03@bk.ru
ORCID iD: 0009-0009-9031-6925
Russian Federation

Ekaterina A. Elpaeva

Almazov National Medical Research Centre; Smorodintsev Research Institute of Influenza

Email: elpaevak@yandex.ru
ORCID iD: 0000-0001-8271-0003
SPIN-code: 8201-1590
Russian Federation

Olga A. Dobrovolskaya

Smorodintsev Research Institute of Influenza

Email: dobrovolskaya.od@gmail.com
ORCID iD: 0000-0001-6654-1107
SPIN-code: 2915-5173
Russian Federation

Elena V. Shmendel

MIREA - Russian Technological University,

Email: elena_shmendel@mail.ru
ORCID iD: 0000-0003-3727-4905
SPIN-code: 7961-5774
Russian Federation

Michael A. Maslov

MIREA - Russian Technological University

Email: mamaslov@mail.ru
ORCID iD: 0000-0002-5372-1325
SPIN-code: 6451-6580
Russian Federation

Yana A. Zabrodskaya

Almazov National Medical Research Centre; Peter the Great Saint-Petersburg Polytechnic University; Smorodintsev Research Institute of Influenza

Author for correspondence.
Email: zabryaka@yandex.ru
ORCID iD: 0000-0003-2012-9461
SPIN-code: 3907-8702

PhD in Physical and Mathematical Sciences

Russian Federation

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Fundings: The study was mainly supported by a grant from the Russian Science Foundation No. 24-15-20026, https://rscf.ru/project/24-15-20026/, together with a grant from the St. Petersburg Science Foundation. Liposome formulation was performed under the financial support of the Ministry of Science and Higher Education of the Russian Federation under the strategic academic leadership program “Priority 2030”, agreement 075-15-2024-221 dated 07.02.2024.


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