Inside our cells, proteins, which are made from RNA, mainly control what our cells do in our body. Now, just imagine the immense power of controlling the production of these proteins using nothing but incredibly tiny molecules. 

siRNA, or small interfering RNA, are short double-stranded RNA molecules that can specifically target certain RNA in cells, preventing the expression of genes in their protein form. Certain cancers and diseases are difficult to treat with conventional drugs, making siRNA molecules useful for targeting the genes involved in such cases. But here’s the catch, delivering these siRNAs into the cytoplasm of cells is no easy task. 

Typically, they have been delivered into the cells by packing them with lipids. But siRNAs are highly unstable, and lipid-based delivery of these molecules cause cytotoxicity, in other words, it causes cells to die. To address this, Rituparna Sinha Roy’s group at IISER Kolkata, in collaboration with Arnab Mukherjee’s group at IISER Pune, turned to molecular engineering to improve the delivery of siRNAs into cells. They sought to increase the stability and reduce the toxicity associated with these molecules. The findings of their research were recently published in Chemical Science.

The researchers designed a lipopeptide-based delivery vehicle for siRNA, utilising protein components instead of relying solely on lipids to transport the molecules. ​“Lipopeptides help carry the siRNA to cells where normal nanoparticles can’t reach,” explained Mukherjee, Professor, IISER Pune.

This delivery vehicle consists of an alternating arginine-sarcosine-arginine (Arg-Sar-Arg) backbone, which comprises two arginine residues separated by a spacer sarcosine (a modified form of the amino acid glycine) residue in the middle. The chosen combination demonstrated optimum protease stability, ensuring that the siRNA remained stable enough to enter the cytoplasm. They also ensured that the siRNA-peptide complex was not overly stable; the siRNA should still separate from its peptide vehicle and be free to do its job of silencing in the cells. 

To further enhance the delivery process, specific types of lipid molecules with unsaturated bonds were incorporated into this vehicle. As a result, the siRNA-lipopeptide complex can rapidly enter the cells, minimising residence time on the cell membrane. This also prevents entrapment inside an endosome, enabling the siRNA to be freely available in the cytoplasm. 

“It is important for the peptide-based transporter to have proteolytic stability to protect the peptide from proteases and also siRNA from RNAses in the cells,” explained Sinha Roy, Associate Professor, IISER Kolkata. But, if the peptide is overly protease-stable, it won’t be cleaved by proteases, and the siRNA will not be free. Our aim is to ensure the internalised cytosolic siRNA remains free, enabling it to participate in the silencing.”

This ​“made-in-India” peptide-based siRNA transporter, for which the scientists have applied for an Indian patent, proves to be more cost-effective. 

It demonstrates better uptake into cancer cells and ​“hard-to-transfect” primary cells compared to the standard lipid-based HiPerFect by Qiagen, commonly used in the field. Upon testing its effects on gene silencing in breast cancer cells, the researchers observed that it effectively silenced key genes implicated in making the cells cancerous with long term efficacy. This indicates that their siRNA transporter holds promising potential as a therapeutic tool for future applications.

“As this is a gene delivery agent, we can deliver nucleic acids through it, which can have many functions,” said Argha Mario Mallick, the first author of the paper. ​“We can utilise this transporter to deliver gene editing tools, develop mRNA-based vaccines, and even reprogram cells via gene therapy.”

Amongst the numerous potential applications in therapeutics, one particularly appealing use is the reprogramming of immune cells in certain cancers. By employing the transporter to deliver gene editing tools like CRISPR-Cas9, immune cells can be modified to effectively target cancer cells. 

Manzoor K., Professor, Amrita School of Nanosciences and Molecular Medicine, Kochi, said, ​“The current method developed by Sinha Roy and her group is promising due to its excellent biocompatibility, non-immunogenicity, and high transfection efficiency”.