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

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Abstract

BACKGROUND: The delivery of nucleic acids to mesenchymal stem cells, which are used as model objects in in vitro experiments or as therapeutic agents in regenerative medicine and oncology, is an actively developing area of research. Existing non-viral delivery systems either have low effectiveness or highly toxic to mesenchymal stem cells. Therefore, the development of new carriers has become an urgent priority.

AIM: To demonstrate the feasibility of delivering model messenger RNA and small interfering RNA to rat heart mesenchymal stem cells (MSCs) in vitro using original cationic liposomes 2X3-DOPE (1:3 molar ratio) and to evaluate the influence of exosomes incorporated into hybrid nanoparticles with 2X3-DOPE on the efficiency of RNA delivery.

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, and hydrodynamic diameter measurement using dynamic light scattering. Small interfering RNA was chemically synthesized; whereas messenger RNA was obtained by in vitro transcription. Complexes of liposomes or hybrid nanoparticles with RNA were prepared by mixing; the properties of the resulting particles were assessed using dynamic light scattering and atomic force microscopy. To evaluate the efficiency of RNA delivery to rat heart mesenchymal stem cells derived from both healthy and ischemic myocardium, we used fluorescence microscopy, laser scanning confocal microscopy, and flow cytometry.

RESULTS: Complexes of cationic liposomes 2X3-DOPE (1:3 molar ratio) with messenger RNA and 2X3-DOPE modified with DSPE-PEG2000 (0.62 mol%) complexed with small interfering RNA were successfully prepared and characterized. It was demonstrated that 2X3-DOPE is ineffective for messenger RNA delivery to rat cardiac mesenchymal stem cells; whereas hybrid nanoparticles incorporating exosomes based on these liposomes exhibited up to 40% transfection efficiency. In addition, 2X3-DOPE modified with DSPE-PEG2000 (0.62 mol%) was effective for small interfering RNA delivery to rat cardiac mesenchymal stem cells, achieving up to 90% transfection efficiency; whereas the use of hybrid nanoparticles based on this formulation resulted in 100% transfected cells with more than a twofold increase in small interfering RNA in the cells as indicated by the average fluorescence intensity.

CONCLUSION: Cationic liposomes 2X3-DOPE (1:3 molar ratio) modified with DSPE-PEG2000 (0.62 mol%) are promising vehicles for small interfering RNA delivery to mesenchymal stem cells, both independently and in combination with exosomes. Exosomes integrated in hybrid nanoparticles based on 2X3-DOPE improve the transfection efficiency of both messenger RNA and small interfering RNA in rat cardiac mesenchymal stem cells in vitro.

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, Saint Petersburg; Saint Petersburg; Saint Petersburg

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

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg; Saint Petersburg; Saint Petersburg

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, Saint Petersburg; Saint Petersburg

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

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg; Saint Petersburg

Ekaterina G. Nikitina

Almazov National Medical Research Centre

Email: purrpurr@list.ru
ORCID iD: 0009-0009-0407-3307
SPIN-code: 5903-8336
Russian Federation, Saint Petersburg

Aleksandr S. Klochev

Saint Petersburg State University

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

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

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg; Saint Petersburg

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, Saint Petersburg

Elena V. Shmendel

MIREA – Russian Technological University

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

Cand. Sci. (Chemistry)

Russian Federation, Moscow

Mikhail A. Maslov

MIREA – Russian Technological University

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

Dr. Sci. (Chemistry)

Russian Federation, Moscow

Yana A. Zabrodskaya

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

Author for correspondence.
Email: yana@zabrodskaya.net
ORCID iD: 0000-0003-2012-9461
SPIN-code: 3907-8702

Cand. Sci. (Physics and Mathematics)

Russian Federation, Saint Petersburg; Saint Petersburg; Saint Petersburg

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Supplementary files

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2. Fig. 1. Characterization of extracellular vesicles isolated from human serum. a, Western blot analysis using antibodies against the specific exosome marker protein HLA class I. M, the molecular weight marker with electrophoretic mobility in kDa shown on the right. EXO, the sample under investigation; the arrow indicates the position of the detected protein band. b, Electron micrograph of a sample of extracellular vesicles. The scale bar is 100 nm long.

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3. Fig. 2. Hydrodynamic diameter of the studied samples determined by the DLS method. EXO, exosomes; LP, cationic liposomes (2X3-DOPE or 2X3-DOPE-PEG for mRNA or siRNA, respectively); EXO-LP, hybrid nanoparticles containing 2X3-DOPE or 2X3-DOPE-PEG; LP/RNA, complexes of liposomes with RNA; EXO-LP/RNA, complexes of hybrid nanoparticles with RNA.

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4. Fig. 3. Surface topography of samples containing 2X3-DOPE/mRNA and 2X3-DOPE-PEG/siRNA complexes (left column), exosomes (middle column), and their hybrids (right column) visualized by atomic force microscopy. A pseudo-color scale bar reflecting the particle height in nanometers (nm) is shown to the right of all images. The scale bar in each image is 500 nm long.

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5. Fig. 4. Transfection efficiency of iCMC and hCMC with complexes of cationic liposome 2X3-DOPE with mRNA-eGFP and complexes of hybrid nanoparticles EXO-2X3-DOPE with mRNA-eGFP, 24 hours post-transfection, assessed by flow cytometry. The statistical significance of differences was determined using one-way ANOVA adjusted for multiple comparisons (Tukey test) (***p < 0.0002, **p < 0.0021).

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6. Fig. 5. Transfection efficiency of iCMC and hCMC with cationic liposome complexes 2X3-DOPE-PEG containing siRNA-JOE and hybrid nanoparticle complexes EXO-2X3-DOPE-PEG with siRNA-JOE, 24 hours post-transfection, assessed by flow cytometry. Statistical significance of the differences was determined using a one-way ANOVA adjusted for multiple comparisons (Tukey test) (**p < 0.0021, *p < 0.0332).

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7. Fig. 6. Transfection efficiency of iCMC (top) and hCMC (bottom) with cationic liposome complexes 2X3-DOPE-PEG containing siRNA-JOE (left) and hybrid nanoparticle complexes EXO-2X3-DOPE-PEG with siRNA-JOE (right), 24 hours post-transfection, assessed by fluorescence microscopy. Cell nuclei are stained with Hoechst 33342 (blue); siRNA is labeled with JOE (yellow). The scale bar is 150 μm long.

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8. Fig. 7. Evaluation of internalization of the studied complexes in iCMC (top) and hCMC (bottom), 24 hours post-transfection, by laser scanning confocal microscopy. Nuclei are stained with DAPI (blue), siRNA is labeled with JOE (green), and the actin cytoskeleton is stained with Alexa Fluor 680 Phalloidin (red). The scale bar is 50 μm long.

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