Chitosan as a supporter for pCRISPR transfer

CRISPR/Cas is a promising tool to tackle genetic diseases. However, non-viral vectors with good transfection efficiency often struggle with high cytotoxicity. In the presented paper, calcium nanoparticles were modified with chitosan. The effects on transfection efficiency and cytotoxicity were then investigated.

CALCIUM-BASED NANOMATERIALS AND THEIR INTERRELATION WITH CHITOSAN: OPTIMIZATION FOR PCRISPR DELIVERY

Calcium‑based nanomaterials and their interrelation with chitosan:optimization for pCRISPR delivery, Rabiee, N.; Bagherzadeh, M.; Ghadir, A.M.; Kiani, M.; Ahmadi, S.; Jajarmi, V.; Fatahi, Y.; Aldhaher, A.; Tahriri, M.; Webster, T.J.; Mostafavi, E., 2021, Journal of Nanostructure in Chemistry, https://doi.org/10.1007/s40097-021-00446-1

CRISPR/Cas is considered a promising tool to combat genetic diseases. To introduce CRISPR and the gene to be transferred into a cell as a plasmid (pCRISPR), a carrier or vector is required. In addition to viral vectors, which are both expensive and could trigger immunological reactions in vivo, non-viral vectors are an alternative. These are often inexpensive to synthesize and produce. They also offer much scope for optimization to enable applications in disease prevention, diagnosis and treatment, for example. However, in previous studies, non-viral vectors showed a correlation between cytotoxicity and transfection efficiency. Vectors with high transfection efficiency, such as the synthesized polymer PEI (polyethyleneamine), were often associated with relatively high cytotoxicity. In contrast, nanoparticles containing cations such as Mg²⁺, Ca²⁺, Ba²⁺, or Mn²⁺ exhibited low cytotoxicity but also a low transfection rate.

Nanoparticles such as calcium phosphate (CaP) are able to form stable complexes with the DNA backbone and additionally stabilize it. During transfection, the nanoparticle-DNA complexes can be introduced into cells via ion channels. However, after introduction into the cell, the exogenous DNA is rapidly degraded, which is why a low transfection rate of the cells is observed here.

Interesting results have also been obtained for the use of natural cationic polysaccharides containing glucosamine subunits, such as chitosan, as vectors. The unacetylated form can form stable complexes with the DNA to be inserted. If the chitosan is smaller than 90 nm, it is able to effectively condense DNA.

To improve the transfection efficiency of calcium nanoparticles, CaP and calcium nanoparticles (CaNPs) prepared in plants were modified with chitosan in the presented study. Subsequently, the chitosan nanoparticles were used to introduce pCRISPR coupled with EGFP into HEK-293 cells. In addition to determining the transfection efficiency by EGFP fluorescence, the cytotoxicity of the vectors was investigated.

RESULTS

• Chitosan-treated nanoparticles exhibited higher zeta potential, resulting in enhanced binding of genetic material to the particles
• All nanoparticles showed good cell viability for HEK-293 cells in assays (more than 85%)
• Transfection efficiency improved with increasing ratios between nanoparticles and pCRISPR DNa
• Best EGFP expression of 25% of HEK-293 cells was observed for CaP-chitosan, and more than 14% for CaNPs-CaP-chitosan 
• Improvement of DNA condensation due to synergistic effect between CaP and chitosan.
• Lower aggregation of nanoparticles in the presence of chitosan resulted in smaller particle size and thus improved transfection

Summary: In this study, it was demonstrated that chitosan-coupled CaP and CaNP nanoparticles are capable of transfecting human HEK-293 cells with pCRISPR. In addition to transfection efficiency up to 25% for CaP-chitosan, good cell viability above 85% was also observed with these non-viral vectors. Link to article: https://link.springer.com/article/10.1007/s40097-021-00446-1