Small interferring RNAs (siRNAs) can inactivate viruses by so-called gene silencing. In order to use this process in biomedicine, the transport of unstable siRNAs must be improved. In the presented study, siRNAs are coupled to chitosan microparticles and investigated for the inactivation of viral proteins in HIV-1 infected C8166 cells. siRNA transfection mediated by chitosan microparticles for the treatment of HIV-1 infections of human cell lines
siRNA TRANSFECTION MEDIATED BY CHITOSAN MICROPARTICLES FOR THE TREATMENT OF HIV-1 INFECTION IN HUMAN CELL LINES
Chronopoulou, L.; Falasca, F.; Di Fonzo, F.; Turriziani, O.; Palocci, C. siRNA Transfection Mediated by Chitosan Microparticles for the Treatment of HIV-1 Infection of Human Cell Lines. Materials 2022, 15, 5340. https://doi.org/10.3390/ma15155340
One of the most promising trends in biomedicine is the evolution from classical drug delivery to the manipulation of gene expression. Small interferring RNAs (siRNA) are able to inactivate certain genes and subsequently prevent the expression of a specific protein. This process occurs through a process known as RNA interference (RNAi). Through the post-transcriptional gene silencing mechanism, plants or fungi, for example, can inactivate viruses. RNAi can also lead to antiviral immunity in mammals. So-called RNAi-based therapeutics therefore offer new possibilities to treat diseases. However, the transport of siRNAs prevents their widespread application. Due to their relatively high molecular weight (approximately 13 kDa) and strong negative charge, siRNAs have difficulty diffusing across the cell wall. In addition, they are unstable in the bloodstream and can trigger immune responses if unprotected.
To enhance the transport of siRNAs, viral and non-viral vectors can be used. Compared to viral vectors, non-viral vectors offer higher safety, simpler synthesis, lower immunity, and improved cell and tissue targeting through specific modifications. Chitosan (CS) and PEI are considered the most promising non-viral vectors. Both have the advantage of being able to form complexes with negatively charged nucleic acids due to their positive charges. This stabilizes the siRNA and also facilitates transport across the cell membrane. Moreover, by adapting the polymers, the physiochemical properties can be optimized for siRNA transport. CS as a non-viral vector has high biocompatibility, good biodegradability, is non-toxic and exhibits comparatively low immunogenicity.
The aim of the study presented here is to design an efficient transport system based on CS microparticles for siRNA to inactivate the HIV-1tat gene in C8166 cells. In this regard, the cytotoxicity, cell uptake depending on the N:P ratio and chemical structure are first investigated using CS/hsDNA microparticles as a model. Then, the experiments are repeated with CS/siRNA-tat/revHIV-1 particles. The synthesized CS microparticles are compared with PEI and commercial liposomes for siRNA transport.
- Comparison of 50 kDa and 150 kDa CS with three different N:P ratios (5, 10, 20), higher particle diameter for 50 kDa CS, this increases with N:P ratio
- No influence of molecular weight and N:P ratio on PDI, zeta potential is more positive at 50 kDa and higher N:P ratios
- Selection of 50 kDa CS with N:P ratio of 10 for further experiments
- CS/hsDNA microparticles exhibited a spherical shape and an average size of 173 nm
- Inclusion efficiency of more than 80%, detection of hsDNA binding of CS by gel electrophoresis
- In vitro release kinetics: first 4 h no more than 11%, after 48 h reach plateau value of 40% → slow and steady release
- CS/siRNAtat/rev-HIV-1: smaller microparticles, higher PDI and larger zeta potential compared to CS/hsDNA, similar entrapment efficiency
- Cytotoxicity: regardless of concentration used (100, 20 and 10 μg/mL), no effect on cell metabolism after 24, 48, and 72 h, although morphological changes were evident
- Complete endocytosis after 4 h
- Infection reduction study: compared to PEI and the liposomes, CS was the only polymer able to achieve significant viral inhibition pre- and post-infection with HIV-1 (40 and 60%)
Summary: In the presented study, siRNA was successfully coupled to CS microparticles and entrapped. The use of CS with an intermediate molecular weight and N:P ratio was the optimal. Overall, this successfully inactivated viral proteins in HIV-1 infected C8166 cells, especially in the post-infection state.
Link to article: https://www.mdpi.com/1996-1944/15/15/5340/htm