Polymeric micro- and nano-carriers as a universal platform for delivery of biologically active substances to therapeutically cell populations
Albert R. Muslimov1,3,5, Tatyana V. Mashel6, Oleksii O. Peltek6, Mikhail A. Trofimov5, Igor S. Sergeev5, Yana V. Tarakanchikova4,5, Alexander A. Goncharenko5, Kirill V. Lepik1, Mikhail V. Zyuzin6, Alexander S. Timin1,2,3
1 Pavlov First Saint Petersburg State Medical University, St. Petersburg, Russia
2 RASA Center in Tomsk, Tomsk Polytechnic University, Tomsk, Russia
3 Peter The Great Saint Petersburg Polytechnic University, St. Petersburg, Russia
4 Optoelectronics and Measurement Techniques Laboratory, University of Oulu, Oulu, Finland
5 Saint Petersburg Academic University, St. Petersburg, Russia
6 Department of Nanophotonics and Metamaterials, ITMO University, St. Petersburg, Russia
Contact: Dr. Albert R. Muslimov
Gene therapy is one of the most perspective methods of the treatment for a number of hereditary, infectious and oncological diseases. Recently this therapeutic approach has received a new development through the discovery of genome editing tools which have great potential due to their high specificity. However, the absence of safe and effective methods to delivery genetic constructs inside relevant cells is a key limitation for the wide application of this technology in clinical practice. Viral vectors have already been applied in medical practice, but there are several limitations associated with their use, such as immunogenicity, mutagenesis, inflammatory response. The need to comply with specific technical requirements also determine the high cost of the final product. Thus, the development of new non-viral intracellular genetic materials delivery tools is an urgent task. Recently, polyelectrolyte micro- and nanocapsules have been considered as one of the promising carriers for the safe and effective delivery of biologically active compounds. The use of biodegradable nanocapsules provides many advantages compared to other delivery systems: high loading capacity, the relatively cheap manufacturing, low toxicity, and the ability to protect the transferred material from the aggressive effects of biological environments of the body. This work aimed to study the effectiveness of the polyelectrolyte capsules as a platform for genetic material delivery.
Materials and methods
The capsules were prepared by layering of oppositely charged of Polyarginine/Dextran sulfate polymers (PARG/DEXS) using Layer-by-Layer technology on calcium carbonate core obtained by co-precipitation of sodium carbonate and calcium chloride aqueous solutions. The following genetic constructs were used: plasmid DNA and messenger RNA, encoding green fluorescent protein (GFP), messenger RNA, encoding TALEN nuclease which causes the deletion of the dTomato gene, and small interfering RNAs (siRNAs) that inhibit the synthesis of GFP. For evaluate the effectiveness of the genetic material delivery by means of polyelectrolyte capsules HEK293T dTomato, HeLa, Mk4 cell lines, and primary cell cultures of human bone marrow mesenchymal stromal cells (hBMSCs) and macrophages were used.
During the research intracellular genetic material delivery platform at the form of polyelectrolyte capsules with a size 300-500 nm have been developed. Such carriers showed low cytotoxicity (viability of more than 90%) in case of using 25:1 capsule to cell ratio. The transfection efficiency of HEK293T dTomato and hBMSCs was 70% for messenger RNA and 40% for plasmid DNA encoding GFP. In the experiment with mRNA delivery to primary human macrophages, transfection efficiency was 60%. Upon transfection of HEK293T dTomato with messenger RNAs encoding TALEN nuclease, knockout of the dTomato gene was observed in 70% of cells. In an experiment with siRNA, suppression of GFP synthesis was detected in 98% of HEK293T HeLa and MK4. For macrophages, mRNA transfection efficiency was about 50%.
We demonstrated that polyelectrolyte capsules are a highly effective and safe platform for in vitro genetic material delivery to the cells. In the future, it is planned to conduct in vivo experiments to study the opportunity of polyelectrolyte capsules usage for the genetic material delivery.
This work was supported by the Russian Foundation for Basic (scientific project No. 19-015-00098). Alexander S. Timin also thanks for support the RFBR (grant No. 18-015-00100). Kirill V. Lepik also thanks for support the RFBR (grant No. 19-29-04025).
Polymeric capsules, non-viral delivery systems, cells transfection, nucleic acids, bioactive substances, encapsulation.